IGFBP7) were overexpressed using lentiviruses. IGFBP7 expression was knocked down with small interfering RNA. The effect of IGFBP7-AS1 in vivo was confirmed by animal experiments. The effect of IGFBP7 on SHED odontogenic differentiation was assessed with alkaline phosphatase staining, alizarin red S staining, quantitative reverse transcription-PCR, and western blotting. The relationship between IGFBP7-AS1 and IGFBP7 was confirmed by quantitative reverse transcription–PCR and western blotting. IGFBP7-AS1 promoted SHED odontogenesis in vivo, and regulated the expression of the coding gene IGFBP7 positively. Inhibiting IGFBP7 led to suppress SHED odontogenic differentiation while IGFBP7 overexpression had the opposite effect. IGFBP7-AS1 enhanced the stability of IGFBP7. IGFBP7-AS1 promoted SHED odontogenic differentiation in vivo. The underlying mechanism may involve the enhancement of IGFBP7 stability. This may provide novel potential targets for dental tissue engineering.Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. We identified the effect of the long noncoding RNA insulin-like growth factor-binding protein 7 antisense RNA 1 (lncRNA IGFBP7-AS1) in vivo and its underlying mechanism during SHED odontogenic differentiation. IGFBP7-AS1 and insulin-like growth factor-binding protein 7 ( Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells in tissue engineering because of their high proliferation ability and fewer ethical issues . As one Zeb2 and downregulated the expression of epithelial cadherin by blocking the Zeb2 5′ untranslated region (5′UTR) splice site [Uchl1 mRNA and affecting short interspersed nuclear elements [Natural antisense transcripts (NAT) are endogenous transcripts complementary to other transcripts in organisms . They caice site . The antelements . IGFBP7-elements .IGFBP7 expression levels by enhancing IGFBP7 stability. In summary, we identified the effect of IGFBP7-AS1 in vivo and its mechanism during SHED odontogenic differentiation.In the present study, we confirmed the odontogenic effect of IGFBP7-AS1 in vivo via animal experiments. Mechanistically, IGFBP7-AS1 regulated http://www.kqgxb.com) and were from children aged 5–7 years. The SHED were cultured as previously described [SHED were kindly provided by Oral Stem Cell Bank  to upregulate IGFBP7-AS1 levels and lentiviruses (MOI: 50) to upregulate IGFBP7 levels. The lentivirus medium contained polybrene (5 mg/mL) to improve the infection efficiency. In addition, the medium was changed after 8 h.EF-1aF/GFP and Puro lentiviruses were constructed by GenePharma  to induce IGFBP7-AS1 and RNA oligoribonucleotides  were purchased from GenePharma . The sequences of siRNA IGFBP7 were as follows: GGGUCACUAUGGAGUUCAATT (sense), UUGAACUCCAUAGUGACCCTT (antisense). SHED were cultured in 12-well plates prior to transfection. After reaching 60% confluence, the cells were transfected with siRNAs using Lipofectamine 3000 .Caries-free human premolars extracted for orthodontics were obtained. For the in vivo experiments, 2-mm high dentin slices were prepared from the roots of premolars using a hard tissue slicing machine. The root canal was enlarged to prepare the shape of a hollow tube and eliminate the pre-dentin. Before cell seeding and transplantation, the dentin slices were prepared in a sterile tissue culture cabinet. The slices were exposed to ultraviolet light for 30 min, rinsed in phosphate-buffered saline (PBS) three times at 1 min per wash, and immersed in 20 mL 1.25% NaOCl, 17% EDTA, and 0.9% normal saline. After disinfection, the slices were washed in PBS three times.5) were mixed with 1 mg hydroxyapatite (HA), then incubated at 37 °C with 5% CO2 for 3 h.The SHED were cultured with α-minimum essential medium supplemented with 10% fetal bovine serum (control group). In another group, IGFBP7-AS1 was upregulated (IGFBP7-AS1 group) as described above. SHED were detached using trypsin–EDTA and resuspended in PBS. The cells  male mice (CB17) were used as subcutaneous transplant recipients. The groups were as follows: control , and IGFBP7-AS1 (dentin slices with IGFBP7-AS1 overexpression SHED). Six implants were placed per treatment group: two implants per animal on the left and right.The operations were performed under anesthesia achieved by intraperitoneal injection of 10% pentobarbital sodium. Incisions (approximately 0.5-cm long) were made on each side on the dorsal surface of each animal. Subcutaneous pockets were created by blunt dissection, and one dentin slice was placed in the pocket. The cell–HA compounds were placed in the hollow tube of the dental slice. The incisions were closed with surgical sutures.The animals were killed 8 weeks after implantation. The implants were fixed in 4% buffered paraformaldehyde solution overnight at 4 °C. After washing in PBS three times, the implants were transferred to 20% EDTA demineralization solution and decalcified for 2 months. The solution was changed daily.For histology, the tissues were stained with hematoxylin and eosin and Masson trichrome to visualize collagen formation . For immunohistochemistry staining of dentin sialoprotein  and dentin matrix acidic phosphoprotein 1 , a 1:100 dilution of anti-human antibody was used .Total RNA was extracted with TRIzol , and 1 μg total RNA was converted to complementary DNA using a PrimeScript RT Reagent Kit . qPCR was performed as previously described . Each geg. The protein concentration was determined using bicinchoninic acid (BCA) Protein Assay  and each lane was loaded with equal aliquots of total protein (20 μg). The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes , blocked in 5% bovine serum albumin for 2 h, and probed with antibodies against the following at 4 °C overnight: IGFBP7 , DSPP , and β-actin . The membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated anti-rabbit immunoglobulin. Protein expression was detected using a western enhanced chemiluminescence blotting kit .The cells were harvested with protein lysis buffer containing a phosphatase inhibitor . The cell suspensions were centrifuged at 4 °C for 30 min at 12,000 × ALP staining was performed on day 7 of the experiment using an ALP staining kit according to the manufacturer’s protocol . Briefly, the cultured cells were rinsed in PBS and the cell layer was fixed in 4% paraformaldehyde for 30 min. Then, it was washed with distilled water and incubated in an alkaline solution for 10 min at room temperature.SHED were rinsed with PBS three times and fixed in 4% paraformaldehyde for 15 min. Then, they were stained with 0.1% ARS (pH 4.0–4.6) for 20 min. Finally, the reaction was terminated with clean water.SHED and IGFBP7-AS1 overexpression SHED were plated in 6-well plates. The SHED were treated 24 h later with 50 μg/mL α-amanitin and were harvested for PCR at 6, 12, and 24 h post-treatment.t test, and comparisons between > 2 groups were analyzed using one-way analysis of variance followed by Tukey’s post hoc test. All data are expressed as the mean ± SD of three experiments per group, and P < 0.05 was considered statistically significant.Statistical calculations were performed using SPSS 21.0 . Comparisons between two groups were analyzed using an independent two-tailed Student’s We conducted animal transplantation experiments to confirm that IGFBP7-AS1 plays a role in regulating SHED odontogenic differentiation. SHED were divided into control and IGFBP7-AS1 overexpression groups. Figure IGFBP7 , which is the evidence that IGFBP7-AS1 is an upstream regulator of IGFBP7.As IGFBP7-AS1 is located on the strand opposite the coding gene BP7 Fig. A, which ion Fig. B. Next, ion Fig. C. These IGFBP7-AS1, and IGFBP7 expression was knocked down with siRNA. Successfully transfected SHED were cultured in OM and harvested on days 7 and 14. The ALP staining showed that ALP expression levels were decreased by IGFBP7 knockdown and increased by IGFBP7 overexpression  to predict the minimum free energy (MFE) of RNA hybridization [IGFBP7-AS1:IGFBP7 duplexes was − 1448.40 kcal/mol, much less than that of IGFBP7 or IGFBP7-AS1 individually . These results indicate that IGFBP7-AS1 may enhance the stability of IGFBP7. To confirm the bioinformatics prediction, we assessed IGFBP7-AS1 and IGFBP7 stability by blocking new RNA synthesis using α-amanitin and measuring their expression levels during a 24-h period. 18S RNA, a product of RNA polymerase I, was not affected during the period and was used as a control. IGFBP7-AS1 and IGFBP7 expression was downregulated after the α-amanitin treatment. SHED overexpressing IGFBP7-AS1 had increased IGFBP7 mRNA stability compared with other groups , by forming duplex RNA and generating more amyloid-β through a post-transcriptional positive feedback mechanism [BAZ2B, increasing the stability of pre-mRNA BAZ2B and further stabilizing the mature mRNA [IGFBP7. Interestingly, we found that IGFBP7-AS1 was expressed concordantly with IGFBP7 during SHED odontogenic differentiation. Furthermore, IGFBP7-AS1 is the upstream regulator of IGFBP7 mRNA. IGFBP7 has been associated with regions of bone and dentine deposition [IGFBP7 improved SHED odontogenic differentiation in vitro. Exploration of the mechanism of IGFBP7-AS1 regulation of IGFBP7 expression showed that IGFBP7-AS1 and IGFBP7 were partially complementary in the exon 1 region with 96 bp. IGFBP7-AS1 may improve the stability of IGFBP7. Bioinformatics analysis calculating the RNA hybridization MFE [IGFBP7 combination is more stable than that of a single transcript. To verify these results, we added α-amanitin, which blocks new RNA synthesis, to SHED overexpressing IGFBP7-AS1 or IGFBP7. IGFBP7-AS1 improved the stability of IGFBP7 mRNA after α-amanitin treatment. Therefore, we conclude that IGFBP7-AS1 regulates SHED odontogenic differentiation by upregulating the stability of IGFBP7.IGFBP7-AS1 belongs to natural antisense RNAs and has been significantly associated with overall survival in patients with glioblastoma . Naturalechanism . Anotherure mRNA . This suposition . It is aposition , 31. Hertion MFE  showed tIGFBP7. This could provide a therapeutic target in dental regeneration. What’s more, we used lentivirus to up-regulate the expression of IGFBP7-AS1 in SHED; however, lentivirus-based cells cannot be used in regenerative medicine. In the future, we should explore the feasible way to up-regulate the expression of IGFBP7-AS1in SHED to immortalize seed cells.In this study, we verified the effect of IGFBP7-AS1 in SHED odontogenic differentiation in vivo and explored the mechanism of IGFBP7-AS1 regulation thereof by improving the stability of


Besides, the heavy homologous nuclides radium-223 and radium-224 – with similar properties to barium – are suitable alpha-emitters for targeted alpha-particle therapy. However, there is a lack of suitable chelation agents, especially for heavy group 2 metals. The macrocycle calix[4]arene-1,3-crown-6 seems to interact with these metals strongly. Therefore, this ligand and its coordination to the divalent cations of barium, strontium, and lead have been investigated. The complex formation was analyzed by NMR and UV/Vis titration experiments in acetonitrile, and stability constants were determined to be >4 with both methods. It was found that the stability of these complexes increase in the order strontium, barium, and lead. Additional to these investigations, X-ray crystallography, solvent-dependent  via NMR and UV/Vis techniques.The stability constants of the promising ligand calix[4]arene-1,3-crown-6 and divalent metals of radiopharmaceutical interest: lead, barium, and strontium, were determined  The potential of radiometals has more recently been realized and relies on their nature of being easily attached to a biomolecule by implementing a chelating agent.2+, Ba2+, and Pb2+ have been selected, since they provide radionuclides, which possess useful properties for application in radiopharmacy as well as nuclear medicine and are therefore of particular interest.For this work, the divalent cations Sr212Pb has demonstrated significant utility in both in vitro and in vivo systems.4 The radionuclide 212Pb is not only a promising β−-emitter but serves as an in vivo generator for the alpha-emitter 212Bi.5 Alpha-emitters combine a short range with a high linear energy transfer, which results in the relatively high biological effect and cytotoxicity.6 Furthermore, the combination of 212Pb and 203Pb is a matched radionuclide pair for image-guided radionuclide therapy. 203Pb decays by electron capture with a γ-emission of 279 keV (80%) which makes it a promising SPECT (single photon emission computer tomography) imaging agent.7In the field of targeted and pre-targeted radioimmunotherapy, 89Sr in the treatment of advanced metastatic prostate cancer has been examined in numerous clinical trials.8–1189Sr in the form of its dichloride (Metastron) is FDA-approved for radionuclide therapy since 1993 and is used for the palliation of painful osseous metastases.12 The element barium offers the radionuclide 131Ba, which decays by electron capture while emitting suitable γ-rays for diagnostic use. The radionuclide 131Ba could be a promising bone-scanning agent in scintigraphy.13,14 The group of heavy alkaline earth metals can also be expanded to its heaviest homolog. The element radium offers the α-emitters 223Ra and 224Ra. Due to their suitable half-lives of 11.4 d, respectively 3.6 d, and their excellent decay properties (a cascade of 4 × α and 2 × β decays), both are ideal nuclides for the targeted alpha-particle therapy.15 Radium, in the form of [223Ra]RaCl2 (Xofigo), is indicated for the treatment of metastatic castration-resistant prostate cancer (mCRPC) and received marketing approval by the EMA and FDA in 2013.16 Since there is no stable radium isotope, it is not a trivial challenge to investigate its chemistry. However, due to its related chemical behavior, barium can be seen as its non-radioactive surrogate and findings derived from the barium chemistry will most likely apply for radium as well.17–19 All mentioned radionuclides and their properties are listed in Alkaline earth metals belong to the most prominent group of divalent ions, which offer a variety of interesting radionuclides. The utility of 1 The essential requirement for a good chelator is the thermodynamic and kinetic stability of its metal–chelate complex in vivo; this necessitates the design of ligands appropriate for the particular radionuclide.2,20 To compare and evaluate chelators with high complexation ability, the knowledge of the stability constant (log K) is mandatory.Although these nuclides possess suitable properties, the absence of stable bifunctional chelating agents for these radiometals hampers their use for targeted radiotherapy and radioimaging.28,29 Though, there are no remotely suitable chelating agents known for heavy alkaline earth metals.There is a constant search for ligands perfectly adapted to the particular application. Noteworthy, the recently developed DOTA-derivative TCMC -1,4,7,10-tetraazacyclododecane) seems to be promising for Pb.30–34 Depending on the metal, the coordination appears to involve not just the crown ether-oxygen donors, but also the aromatic phenol-units of the calixarene.351H NMR studies can provide further experimental evidence for the influence of cation–π-interactions and are a key part of this work.Calix-crown ethers are a widely investigated class of ligands for cations based on the calixarene backbone. They provide a suitable coordination chemistry towards alkali metal and alkaline earth metal cations.The calix[4]arene-1,3-crown-6 bears two hydroxyl groups located on opposite sides. These groups act as ionizable units by the loss of their protons under basic conditions and thus to form a neutral complex witch a divalent cation. Additionally, the hydroxyl functions can straightforwardly be modified by attaching other ionizable units.32,36,37 Therefore, UV/Vis and NMR titration experiments were established as reliable and constant methods for the determination of the stability constants of calix 1 with Sr2+, Ba2+, and Pb2+. Due to an activity-related concentration limit, Ra2+ could not be investigated.The objective of this research was to evaluate calix[4]arene-1,3-crown-6 (1) as a lead compound that could, upon further modifications, yield a viable ligand for the above mentioned divalent cations. Literature about alkaline earth metal ligands, especially about radium, are mostly focused on extraction studies without information about comparable stability constants.31Two synthetic routes are described in the literature for the preparation of ligand 1, both using the starting material calix[4]arene. A high yield of the ligand 1 can be obtained when first alkylating the calix[4]arene to obtain the 1,3-dimethylcalix[4]arene, followed by the alkylation of the remaining two hydroxyl groups with pentaethylene glycol ditosylate (crown ether moiety) followed by the cleavage of the methyl groups with trimethylsilyl iodide.tert-butoxide is used as a base to deprotonate the hydroxyl groups; additionally the potassium cations serve the purpose of a template effect and arrange the formation of the cone conformation. However, this reaction takes 3 days and obtains a moderate yield.38 For this work, ligand 1 was obtained with a higher yield of 50% using a modified reaction according to the second route.39The second route is a one-pot reaction starting directly with the calix[4]arene and pentaethylene glycol ditosylate. Potassium P2/n. The n glide plane of the space group cuts through the calixarene molecule as well as through the carbon atom of the dichloromethane molecule. Therefore, the asymmetric unit contains only half of both molecules. The part of the crown ether ring starting from O3 up to O3′  scale" fill="currentColor" stroke="none"><path d="M80 840 l0 -40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 120 0 120 0 0 -40 0 -40 -80 0 -80 0 0 -40 0 -40 80 0 80 0 0 -80 0 -80 -120 0 -120 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 120 0 120 0 0 40 0 40 40 0 40 0 0 80 0 80 -40 0 -40 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -120 0 -120 0 0 -40z M720 840 l0 -40 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 80 0 80 40 0 40 0 0 80 0 80 40 0 40 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 -80 0 -80 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 200 0 200 0 0 40 0 40 -80 0 -80 0 0 40 0 40 40 0 40 0 0 80 0 80 40 0 40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -120 0 -120 0 0 -40 0 -40 -40 0 -40 0 0 80 0 80 40 0 40 0 0 80 0 80 40 0 40 0 0 80 0 80 -40 0 -40 0 0 -40z"/></g></svg> − x, y, ½ − z) is disordered with two different orientations as well as the chlorine atom of the dichloromethane molecule. This disorder is refined using split positions (A and B) for each affected atom (group). The different orientations of the ether part and the dichloromethane molecule are shown in When slowly evaporating the solvent of a saturated solution of 1 in dichloromethane, the formation of single crystals was observed. These crystals were analyzed by single-crystal X-ray diffraction, which unfolded an inclusion complex with dichloromethane located in the upper rim of the aromatic calix[4]arene part . Compoun40 The oxygen atoms of the crown ether moiety  and symmetry equivalents) are not primarily directed toward the inner part of the cavity, as they would do whenever a metal cation is coordinated. The diameter of this cavity, as defined by the distances of opposite oxygen atoms, is 5.887 Å  2 times) and 6.849 Å ). The shortest hydrogen bonds are found intermolecularly as O1–H1A⋯O2 contact at a D⋯A distance of 2.767(1) Å and a D–H⋯A angle of 167.7°. All other possible proton contacts have D⋯A distance longer than 3.0 Å.The four aromatic rings of the calixarene form a cavity, which is expanded at the upper rim. The opposite rings are inclined to each other with an angle of 34.5° of the two rings, which are attached to the crown ether moiety, compared to the other two unsubstituted rings, where the angle between the two main planes through the ring carbon atoms was determined with 84.2°. A little above the upper rim, the (disordered) dichloromethane molecule is located. The two hydrogen atoms, whose positions were initially located from the difference Fourier map and later refined using riding models, are oriented such that they have relatively close distances to the centers of the phenyl groups, which are attached to the crown ether residue. The distance of these protons to the center of the rings is 2.485 Å. This is close enough to be discussed as weak C–H–π interaction.41–43 but not particularly for ligand 1, it was necessary to investigate the influence of the solvent on its conformation. Due to their diversion in polarity, size and solvation effects, the solvents CDCl3, acetonitrile-d3, benzene-d6, DMSO-d6, and acetone-d6 have been selected, and the interaction with ligand 1 was studied by 1H NMR measurements , proving the cone conformation of the calix crown .45 The distance between H6 and H7 indicates the angle of the aromatic rings. If the rings are facing each other, the ΔδH7–H6 value should be in the range of 0.9 ± 0.2 ppm,45 which applies to all spectra, but not to benzene (ΔδH7–H6 = 1.3).Due to the results derived from the X-ray spectroscopy, it had to be assumed that the solvent has a strong influence not only on the solid-state conformation but also on the conformation of ligand 1 in solution. Since the incorporation of small molecules and solvent molecules into calixarenes has been widely discussed in the literature in general,urements . The sam2J), and except of benzene-d6, the signals in the spectra are similar arranged for the different solvents. As expected, there is an influence of the solvent on the hydroxyls (H1), depending on its polarity and amount of acceptor sites. The stronger the hydroxyl oxygen can coordinate to the solvent molecules, the less electron density remains at the H1 proton (downfield shift). However, this does not apply for the solvent benzene, which has the lowest polarity in comparison, but a significant H1-downfield shift. A likely explanation for the solvent effect of benzene on the calix 1 is illustrated in No significant influence of the solvent was observed when comparing the coupling constants from The other smaller solvents, as represented in the crystal structure , instead3 is superior over DMSO-d6, because DMSO is known to form complexes with divalent cations.39,46 For these reasons and since the compound is not soluble in water, the stability constants are determined in acetonitrile. Two different methods have been used to determine the stability constants: NMR and UV/Vis titration with the respective metal perchlorate salts.Comparing the different spectra, the signals received from the acetonitrile-measurement, are sharp and well separated, and there does not seem to be significant interaction with the hydroxyl functions. Additionally, we use metal perchlorate salts to study the ligand–metal-interaction; those are highly soluble in acetonitrile. The use of acetonitrile-d1H NMR signals were observed, until the 1:1-complex was completely formed. When comparing the 1H NMR spectra of Ba2+-, Sr2+-, and Pb2+-1:1-complexes  to 0.5 ppm (1:1-complex), caused by the contraction of the lower rim .Stability constants of these complexes were determined by the relationship between the shifts of a selected proton signal and the equivalents of the cation. A reliable method to determine the stability constants 1H NMR chemical shifts of the signal H4 against the equivalents of Ba2+  has a medium sensitivity NMR spin-½ nucleus.48,49 For this experiment, lead(ii) perchlorate trihydrate was dissolved in acetonitrile-d3 and titrated with ligand 1. The difference between the 207Pb signal of Pb(ClO4)2 and Pb-1 resulted in a chemical shift of Δδ = 296 ppm  against the equivalents of Ba2+, 2+, Pb2+, Na+, and Bu4N+ were carried out comparably  have been performed and the stability constants were determined by NMR measurements in acetonitrile-d3 as well as by UV/Vis measurements in acetonitrile revealing log K values for all divalent metals of >4  using TMS as an internal standard. All 207Pb spectra were recorded at a frequency of 125.1 MHz on an Agilent DD2-600 MHz NMR spectrometer with ProbeOne at 298 K using a 90° pulse width of 6.0 μs, a 0.157 s acquisition time, and a 0.6 s delay time. A 1.0 M Pb(NO3)2 solution  was used as an external standard . Calix[4]arene , pentaethylene glycol di(p-toluenesulfonate) , potassium carbonate anhydrous , acetonitrile , dichloromethane , hydrochloric acid , and sodium sulfate anhydrous  were used as obtained for the synthesis of ligand 1. Preparative column chromatography was carried out with silica gel 60 , petroleum ether , and ethyl acetate . For the preparation of the complexes, barium perchlorate , sodium perchlorate , and strontium perchlorate  were stored at room temperature under vacuum and used without further purification. Lead(ii) perchlorate trihydrate  was used as obtained. The solvents used for NMR measurements were purchased from Deutero GmbH.tert-butyl derivative.39 Briefly, a suspension of calix[4]arene , pentaethylene glycol di(p-toluenesulfonate)  and K2CO3  in acetonitrile (100 mL) was refluxed under argon for 7 days. After cooling to rt, the solvent was removed and dichloromethane (50 mL) was added. The suspension was washed with 10% HCl (2 × 50 mL) and water (1 × 50 mL). The organic layer was dried over Na2SO4. After evaporation of dichloromethane, the crude mixture was purified by column chromatography . The product was obtained as a colorless solid . Analyses were in accordance with the previously published literature.38The synthesis of 1 was done according to the procedure of the respective 6 (2.0 × 10−3 M) and 1.0 mL was pipetted in an NMR tube. The sample was referenced to the residual solvent signal. Then, the complexation of cations with 1 was studied. A 0.1 M solution of the metal perchlorate was prepared in the same solvent. Next, stepwise portions (2 μL) of the respective perchlorate solution were added into the NMR tube containing the ligand, and after extensive mixing, the complexation-induced shifts were recorded. At a ligand : metal ratio of 2 : 3, 30 μL portions of a 1.0 M perchlorate salt solution were used, and stepwise additions were continued until a ligand : metal ratio of 1 : 6 was reached to exclude the formation of a complex with another stoichiometry. The displacements of selected 1H NMR signals of ligand 1 upon addition of the perchlorate salt were used to calculate the complex stability constants. All calculations were performed using the WinEQNMR2 software.47 The advised range for the data input covers the addition of metal to ligand from 0.1 to 0.9 equivalents. This instruction was followed and 9 points in this range were measured (steps of 0.1 equiv.) and used for the calculation. The formation of a 1:1 complex was proven by plotting the changes of selected signals against the cation to chelate ratio, observing the change of the slope at a ligand : metal ratio of 1 : 1.A solution of 1 was prepared in acetonitrile-dii) perchlorate trihydrate in acetonitrile-d3 (5.0 × 10−2 M) and a solution of ligand 1 in acetonitrile-d3 (1.0 × 10−1 M) were prepared. A 1.0 mL aliquot of the lead solution was pipetted in an NMR tube. The sample was measured and the 207Pb NMR signal determined. Next, stepwise portions of ligand 1  were added into the NMR tube containing the lead solution, and the spectra were recorded after extensive mixing.A solution of lead(4)2 solution in acetonitrile. The absorption spectra were measured in the range of 250 to 400 nm. The calculation of the stability constants was accomplished using the HypSpec 1.1.18 program.54A solution of ligand 1 was prepared in acetonitrile. The concentration range was chosen to be 0.15 mM, depending on the absorption maximum of the compound. This solution (2.5 mL) was pipetted into a quartz cuvette of 1 cm optical path length. The titration was performed by stepwise addition of a 15 mM Ba(ClOλ = 0.71073 Å). The measurements were performed at −150 °C. The structure was solved by direct methods and refined against F2 by full-matrix least-squares using the program suites from G. M. Sheldrick. All non-hydrogen atoms were refined anisotropically; all hydrogen atoms were placed in geometrically calculated positions and refined by using riding models.55–57 CCDC 1950592 contains the supplementary crystallographic data for the title compound.†Diffraction data were collected using a Bruker Nonius Apex Kappa-II CCD diffractometer with graphite-monochromated MoKα radiation (There are no conflicts to declare.RA-009-C9RA07293D-s001RA-009-C9RA07293D-s002


Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in  Transcrpression .In plants, lncRNAs are implicated in numerous biological mechanisms with demonstrated functions in flowering, organogenesis, photomorphogenesis, reproduction, and abiotic/biotic stress responses . We find that the COBRA lincRNAs display germination- and developmental-dependent patterns of abundance and, in particular, we focus on lncCOBRA1 which contains two snoRNA sequences within it, indicating the first evidence of a sno-lincRNA in Arabidopsis. Unlike sno-lncRNAs identified in humans, lncCOBRA1 is transcribed from an intergenic region, and is transcribed as a longer transcript before processing at its 3′ end to a final length of ∼500–600 nt. We further show that lncCOBRA1 influences plant germination and growth, as plants lacking lncCOBRA1 germinate later and are smaller than wild type plants. Lastly, we identify lncCOBRA1-interacting proteins, including the scaffold protein RACK1A, and several of its known interactors and hypothesize that lncCOBRA1 functions with RACK1A to affect ribosome biogenesis.Here, we assess the function of those nuclear, protein-bound, and conserved lincRNAs that we have termed Brassica rapa than those lacking RBP binding sites (Brassica rapa and nuclear protein binding suggested that these lincRNAs may have important functions in plant systems and were named CONSERVED IN BRASSICA RAPA 1-14 (lncCOBRA1-14)  interaction sites . These png sites . Since lng sites . Thus, tBRA1-14) .lncCOBRAs and initiated our search for function by examining their abundance profiles during seed germination, as lincRNAs in several eukaryotic species are essential during development   of lncCOBRA transcripts displayed germination-dependent patterns of abundance, with peaks in abundance at various points during seed germination , as they had the highest abundance in 2-day-old seedlings and rapidly decreased in abundance as the seedlings aged (We selected a set of these COOLAIR) . Using aCOOLAIR) , we founmination . Going fnditions . Abundaneedlings . Supporteedlings  revealedbibition . The abungs aged .lncCOBRA transcripts displayed tissue specific patterns of accumulation. For instance, we found that lncCOBRA5 abundance is highest in leaf tissue and increases in abundance as the age of the leaf progressed from embryonic cotyledons to juvenile leaves and adult leaves, while lncCOBRA3 demonstrated similar levels of abundance in all tissues profiled  decrease in abundance between 5-day-old cotyledons and true leaves (both juvenile and adult leaves)  . Thus, alncCOBRA1, lncCOBRA3, and lncCOBRA5 as well as nuclear (U6) and cytoplasmic (5.8S rRNA and 18S rRNA) positive controls. All three lncCOBRA transcripts were significantly  enriched in the nuclear fraction like U6 but not in the cytoplasmic fraction where the two rRNAs were enriched, confirming these transcripts were indeed primarily nuclear localized  technique , 2011 anocalized .lncCOBRA1 and lncCOBRA3 contain small nucleolar RNA (snoRNA) sequences annotated within their transcripts  and AT1G05907 (sno-COBRA1B) having ∼79 and 56% sequence identity among the profiled species, respectively , which also included sno-COBRA1B  amplifying the region between the two snoRNAs  to determine the 5′ end of the transcript. In 5′ RACE, any present 5′ caps are removed, and an adapter is directly ligated to the 5′ end of RNA. Following reverse transcription with a gene specific primer and two rounds of PCR, the precise 5′ end of the transcript can be determined  suggests that lncCOBRA1 is not polyadenylated in its final processed form.To determine the precise 3′ end of sted PCR . The finE primer , and theE primer  indicaten length , and is Telo- box and a Site II element (combined referred to as TeloSII)  . Notablyromoters . This coogenesis . Interesogenesis . In addiogenesis , which aogenesis . The prene locus . OveralllncCOBRA1, we obtained a T-DNA insertion line  from the Arabidopsis Biological Resource Center with an insertion upstream of sno-COBRA1A and generated a complete lncCOBRA1 null (lnccobra1-2) using CRISPR gene editing  depleted levels of lncCOBRA1 as measured by qPCR and lnccobra1-2 levels were unmeasurable as it is a null mutant with the entire gene deleted . lncCOBRA1 complementation resulted in a significant increase in lncCOBRA1 levels , suggesting that the slight increase in abundance of these mature rRNAs may in fact be due to the loss of lncCOBRA1  fewer lnccobra1-1 and lnccobra1-2 seeds germinated than in the Col-0 background, while significantly  more lncCOBRA1/lnccobra1-1 seeds germinated at 48 h  delayed in leaf production compared to same aged Col-0 plants. This same trend was also observed in lnccobra1-2 plants, but not to a level of statistical significance    rescued this phenotype and resulted in plants that were slightly larger in both 3- and 5-week-old plants  . Increas t-test) , suggestto Col-0 . lnccobrd plants . Aside fd plants . Since af growth , it is plncCOBRA1, we set out to identify what proteins bind lncCOBRA1, as lncCOBRA1 was initially identified for having sites of RBP binding  enriched with probes antisense to lncCOBRA1 compared to the scrambled sequence control probes, indicating that the lncCOBRA1 probes are highly specific  depleted when ChIRP was performed in lnccobra1-2 null seedlings  (lncCOBRA1 probes compared to scrambled sequence control probes (N = 206), and (3) enriched > 2-fold in Col-0 compared to lnccobra1-2 seedlings  , (2) enr N = 74; . A totallncCOBRA1-interacting proteins were significantly enriched for proteins with molecular function of RNA binding, and 37.5%  were demonstrated to bind to RNA in a recent study identifying the RNA binding proteome of Arabidopsis leaves , which has hypothesized roles in rRNA transcription , gluconeogenesis, and photorespiration  more interactions than expected, indicating that lncCOBRA1-interacting proteins had more interactions among themselves than what would be expected for a random set of proteins of a similar size from the Arabidopsis proteome  , which wATARCA)  were identified in at least 2 biological replicates of lncCOBRA1 pulldown in Col-0  . RACK1A d humans . Severald humans , 40 of win Col-0 . This inin Col-0 . Nearly lncCOBRA1 is a ∼500–600 nt lincRNA with germination-, developmental-, and tissue-specific patterns of abundance, with high abundance early during seed germination and decreases as development progresses. Further, we demonstrate that loss of lncCOBRA1 results in delayed cotyledon emergence and overall smaller plants. We demonstrate that lncCOBRA1 interacts with a wide variety of proteins, including many nucleolar proteins and scaffold proteins, including the highly conserved RACK1 subunit RACK1A, leading to an overall hypothesis that lncCOBRA1 acts as a scaffold to bring together proteins involved in several different processes to ultimately regulate plant germination and development.In this study, we use genetic, biochemical, and proteomic analyses to describe a highly conserved, previously uncharacterized sno-lincRNA with functions in seed germination and development. We reveal that CONSERVED IN BRASSICA RAPA 1-14 (lncCOBRA1-14) that were identified for their interactions with nuclear RBPs and sequence conservation in Brassica rapa  in Arabidopsis   . UltimatlncCOBRA sno-lincRNAs described here are also transcribed from intergenic regions throughout the genome. Thus, lncCOBRA sno-lincRNAs represent a previously uncharacterized class of lincRNAs with potentially important biological functions that warrant future studies.In mammals, the majority of functional snoRNAs are encoded within introns and processed from excised and debranched introns by exonucleolytic trimming. Similarly, all identified mammalian sno-lncRNAs are generated from excised introns as well . In ArablncCOBRA1 contains several conserved elements within its promoter known to be present in the promoters of genes involved in ribosome biogenesis and translation. This includes Telo-box and Site II elements (TeloSII) (lncCOBRA1-interacting transcription factor PUR ALPHA-1 (PURα) (PURA) which is a member of the sequence-specific single-stranded nucleic acid-binding Pur family of proteins. The amino acid sequence of Pura is extraordinarily conserved in sequence from bacteria through humans, where it functions as a transcriptional activator, and as an RNA transport protein. While less is known about PURα in Arabidopsis, it was identified to be an RBP  . Interes1 (PURα) . PURα ise an RBP  and was e an RBP . TCP20 ain genes . In Arabromoters . This coogenesis . Thus, track1a rack1b double mutants have a decrease in abundance of the 60S ribosomal subunit and 80S monosomes, but no differences in polysomes, suggesting a role for RACK1 in ribosome biogenesis  . When gr deleted . Overalle to ABA , 2011 ane to ABA , suggestlncCOBRA1 interaction   . RNJ encelopment . While Relopment , it doeselopment . Insteadelopment . While Relopment . Furtherelopment . Thus, ilncCOBRA1. Given its function in ribosome maturation in bacteria and the multiple functions of RNases on ncRNAs described previously, we posit that RNase J may have additional function in sno-lincRNA processing in Arabidopsis, specifically the 3′ end processing we observe for lncCOBRA1, but future studies will be required to support this hypothesis.RNases are essential for non-coding RNA processing and each RNase can have a multitude of targets. For example, RNase P is an endoribonuclease canonically functions to process the 5′ termini of pre-tRNAs but can also cleave other tRNA like structures in the 3′ end of lncRNAs to form mature 3′ ends . AdditiolncCOBRA1 results in growth phenotypes. While future studies are required, we provide evidence that lncCOBRA1 interacts with a plethora of proteins involved in many different processes. Overall, we hypothesize that lncCOBRA1 acts as a scaffold to bring together many different proteins to regulate normal biological processes, including ribosome biogenesis.In total, using transcriptome-wide analyses we identified functional candidate lncRNAs based on sequence conservation and the presence of RBP binding sites. We further show the loss of All plants were of the Columbia-0 ecotype and were grown in controlled chambers with a cycle of 16 h light and 8 h dark at 22°C. All seeds used for plate growth were sterilized in 100% ethanol for 1-min followed by a 10-min wash with 30% Clorox and 0.01% Tween-20 solution and rinsed five times with sterilized water. Seeds were then plated and grown on 1/2 MS agar plates with 1% sucrose and 0.8% Phytoblend and stratified by cold treating at 4°C for 48 h then placed in growth chambers with the parameters noted above.lncCOBRA1 was previously referred to as AT1NC031460 in AT1G05913 in the Araport11 genome annotation. lnccobra1-1 (SALK_086689) was purchased from the Arabidopsis Biological Resource Center and backcrossed once to Col-0, segregated, and homozygous mutants obtained and validated by PCR. RT-qPCR was used to validate significant depletion in the abundance of lncCOBRA1.lnccobra1-2, the suite of plasmids designed for multiplexed CRISPR genome editing by 1 and used to generate Arabidopsis CRISPR-Cas9 transformation vectors  sequence requirement. The Arabidopsis thaliana TAIR10 genome was used to ensure the specificity of chosen guide RNAs. The first guide RNA (protospacer sequence: 5′-TATGATTTGATCATCATCGG-3′) is located approximately 50 base pairs upstream of the AT1G05913 transcription start site, and the second guide RNA (protospacer sequence: 5′-TATATGGCTCTGGAAGAGGG-3′) is located approximately 121 base pairs downstream of the AT1G05913 transcription start site. Complimentary oligos were designed for each protospacer that contained overhangs compatible with the Arabidopsis U6 promoter driven guide RNA vectors designed by To generate  vectors . Two difAT1G05913, the cloning procedures provided by Arabidopsis thaliana (Col-0) using the floral dip method  . T2 plants were genotyped to test for a deletion in  200 bp) , and SanlncCOBRA1 promoter: lncCOBRA1/lnccobra1-1, the entire 1509 bp between the two neighboring genes was amplified from Col-0 genomic DNA and cloned into BspEI and BstEII restriction enzyme sites of pCAMBIA3301. Transgenic plants were obtained and selected as previously described  and further homogenized using QIAshredders . RNA was then isolated using the miRNeasy mini columns as described by the manufacturers’ protocol . Following elution from the miRNeasy column, RNA was treated with RNase-free DNase  for 25 min at room temperature, ethanol precipitated and resuspended in nuclease-free water supplemented with 1.25% RNaseOUT .All reverse transcription (RT) reactions were performed using SuperScript II following the manufacturers’ instructions with 2.5 mM Random Hexamers , 100 units SuperScript II and 30 units RNaseOUT  for 2 min at 25°C, 90 min 42°C, 5 min 95°C, hold at 4°C. Before qPCR, cDNA was diluted 1:10 for all RT-qPCR reactions except for ChIRP in which the RT reaction was diluted 1:5.UBC9 and UBC10. All primers are listed in qPCR was performed with 2X SYBR Green qPCR Master Mix with Rox #2 , as follows per well: 10 μL 2X SYBR Green Master Mix, 1.5 μL cDNA (diluted 1:10), 0.4 μL Rox #2. 2.1 μL water, 6 μL combined 1.5 μM forward and reverse primers. All qPCR reactions were performed in three technical replicates and all primers tested using water to detect background signal and melt curves were analyzed for a single peak. All qPCRs were run using the following program: 95°C for 10 min; 40 cycles of 95°C 30 s, 55°C 30 s, 72°C 30 s. Melt curves were generated by heating the final PCR 1.6°C/s to 95°C for 15 s, decreasing the temperature to 60°C at 1.6°C/s and slowly increasing back to 95°C at 0.1°C/s. Unless otherwise noted, all qPCR experiments were normalized to the geomean of To examine RNA abundance in nuclei and cytoplasmic fractions, seeds ubiquitously expressing a biotin ligase receptor peptide fusion protein that is targeted to the nuclear envelope (UBQ10:NTF/ACT2p:BirA Columbia-0 ecotype) were used , 2011. AlncCOBRA1 abundance. The sample of adult leaves included a mix of rosette leaves older than leaves 1–4 which were denoted juvenile leaves.For the germination time course, seedlings were collected 2, 3, 4, 5, 7, and 10 days after stratification and flash frozen in liquid nitrogen and stored in –80°C for further processing. Tissues from 5-week-old Col-0 plants were collected, flash frozen in liquid nitrogen, and stored in –80°C until processing for examining the tissue specificity of AT1G05913 gene, the entire Arabidopsis cDNA sequence was used as query for BLAST using CoGeBlast3 using default parameters  against representative Brassicaceae species. The top hits for each species were selected based on e-value and quality score and used for subsequent sequence alignments. Selected sequences were aligned using Geneious Prime  in 1X T4 RNA Ligase Buffer  supplemented with 20 units RNaseOUT  in a total reaction volume of 10 μL for 1 h at 37°C and stored at –20°C overnight.Five μg of RNA from 5-day-old seedlings was first treated with 1 unit of Shrimp Alkaline Phosphatase  in 1X SAP buffer provided and supplemented with 1 mM DTT and 60 units RNaseOUT  for 1 h at 37°C. The SAP reaction was inactivated for 15 min at 65°C and the RNA ethanol precipitated overnight. To remove any 5′ mlncCOBRA1 qPCR Reverse set 2”) was added to the ligase reaction and heat treated at 80°C for 3 min followed by 2 min on ice. Reverse transcription was performed with 100 units SuperScript II in 1X First Strand Buffer, 2 mM dNTPs, 10 mM DTT, and 10 units RNaseOUT  for 1 h at 42°C, 10 min 50°C, 15 min 70°C, hold at 4°C and store at –20°C overnight.On the following day, the 5′ adapter was added. To the 10 μL RppH reaction, we added 1 μL of 5′ RNA adapter  that was first heated to 70°C for 2 min followed by 2 min on ice to relieve secondary structures, 1 μL 10 mM ATP , 10 units T4 RNA Ligase 1 , 1 μL T4 RNA Ligase Buffer , and 40 units RNaseOUT  and incubated for 3 h at 20°C followed by an overnight ethanol precipitated. For cDNA synthesis, 1 μL of gene specific primer  with forward primer “reverse transcription primer (RTP),” and reverse primer “lncCOBRA1 5′ RACE Primer 1” with cDNA diluted 1:5 with the following program: 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; 72°C 5 min, hold at 4°C. PCR 2 was performed similarly, but with PCR reaction 1 diluted 1:20 as the template and “Internal RA5 Primer” forward primer and either (A) “lncCOBRA1 qPCR Reverse set 1” or (B) “lncCOBRA1 5’ RACE Primer 2” as the reverse primer. The PCR reaction was then run on a 1% agarose TAE gel with a 1 kb plus DNA ladder . All primers are listed in To ligate the 3′ adapter, the 3′ RNA adapter  was first heated to 70°C for 3 min and snapped cool on ice for 2 min. Eight μL heat-treated 5 μM RA3 was added to 1 μg RNA isolated from 5-day-old Col-0 seedlings and incubated with 200 units T4 RNA Ligase 2, truncated  in 1X T4 RNA Ligase Buffer  for 1 h 15 min at 28°C. As a control, this reaction was also performed in the absence of T4 RNA Ligase 2, truncated (–Lig). The reaction was then ethanol precipitated overnight.The following day, the precipitated RNA was split in half for reverse transcription ± RT. To 8 μL RNA, 1 μL 10 mM dNTPs and 1 μL RTP was added and incubated for 5 min at 65°C then transferred to ice for 2 min. Reverse transcription was performed with 100 units SuperScriptII (SSII) in 1X First Strand Buffer, 10 mM DTT, 20 units RNaseOUT  for 2 min at 25°C, 90 min 42°C, 5 min 95°C, hold at 4°C and store at –20°C overnight. The reaction was also performed without SSII as a control.The first round of PCR was performed using 1X Phusion High-Fidelity PCR Master Mix with HF Buffer  with cDNA diluted 1:10 in water, and forward primer “RTP” and reverse primer “Illumina RNA index primer 35” with the following program: 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; 72°C 5 min, hold at 4°C. PCR 2 was performed similarly, but with PCR reaction 1 diluted 1:20 as the template and “lncCOBRA1 3’ RACE Primer 1” forward primer and “RNA primer index universal” as the reverse primer. The PCR reaction was then run on a 1% agarose TAE gel with a 1 kb plus DNA ladder , excised and gel extracted using the Monarch DNA Gel Extraction Kit following the manufacturers’ instructions .The purified PCR reaction was then A-tailed with 15 units Klenow Fragment (3′–5′ exo-)  in 1X NEB Buffer 2 , and 0.1 mM dATP for 30 min at 37°C. The reaction was then cleaned up using Zymo ChIP DNA Clean & Concentrator following the manufacturers’ instructions . The resulting PCR reaction was then cloned into pGEM T-Vector system and selected for using the XGal/IPTG system . Sanger sequencing was performed at the University of Pennsylvania Genomic Analysis Core with the SP6 promoter/primer. All primers are listed in Gel was performed using NorthernMax reagents . Ten μg of total RNA for each genotype was added to appropriate amount of 3X NorthernMax Formaldehyde Loading dye, boiled at 65°C for 15 min and flash cooled on ice. 0.5 μL of ethidium bromide (10 mg/mL) was added to each sample and loaded onto a 1.5% NorthernMax denaturing agarose gel and run for ∼3 h at 100 V. Gel was visualized by UV.lnccobra1-1, lnccobra1-2, and lncCOBRA1/lnccobra1-1 were sterilized in 100% ethanol for 1-min followed by a 10-min wash with 30% Clorox and 0.01% Tween-20 and washed 5X with sterilized water. Seeds were then plated on 1/2 MS agar plates with 1% sucrose and 0.8% Phytoblend and stratified by cold treating at 4°C for 48 h and placed in growth chambers. Two days after transfer to growth chambers, the number of seeds that that displayed cotyledons entirely emerged from the seed coat were counted. Plates were then allowed to grow for 3 more days and 5-day-old seedlings were collected to measure lncCOBRA1 abundance.For germination experiments, seeds of Col-0, lnccobra1-1, lnccobra1-2, and lncCOBRA1/lnccobra1-1 were grown in soil as described above. Every day at ∼11 AM the presence of leaf primordia was examined. Leaf initiation was measured when the leaf primordia was visible to the eye (∼0.5 mm). After 3 weeks, plants were weighed for fresh weight measurements. To measure plant size, 3-week-old plants were taped flat on paper, scanned, and analyzed using ImageJ as follows. Scanned images were first converted to 8-bit and processed into a binary image such that any plant tissue was converted to white and background became black. Threshold was set using default settings, inverted, and the “particles” (plants) perimeter and area measured. Area of leaf 3 was selected by hard and measured.Col-0, 4 with a 3′ Biotin TEG. 5-day-old Col-0 and lnccobra1-2 seedlings were crosslinked in PBS with 1% formaldehyde (v/v)  added and placed under vacuum for 10 min, followed by a 5-min quench with 125 mM Glycine under vacuum. Crosslinked tissue was then washed five times in distilled, deionized water, patted dry with paper towels, flash frozen in liquid nitrogen, and stored at –80°C until further processing. Chromatin from 6 g of 5-day-old Col-0 and lnccobra1-2 crosslinked seedlings (3 g scrambled probes and 3 g lncCOBRA1 probes) was isolated as previously described  were first chemically treated to protect the streptavidin from tryptic proteolysis in preparation for mass spectrometry to reduce streptavidin signal as previously described .lncCOBRA1 probe sample and 2X Hybridization buffer was added  supplemented with PMSF (100 μL/10 mL), RNaseOUT , and cOmplete Protease Inhibitor Cocktail . 100 pmol of probes were then added per 1 mL chromatin  and the samples incubated in a 37°C hybridization oven with rotation.Chromatin isolation by RNA purification was performed as previously described , 2015, wAfter 5 h, 100 μL of modified beads were added to each tube and incubated in a 37°C hybridization oven with rotation for another 2 h. Samples were then centrifuged for 5 min at 3000 RPM, supernatant removed, and resuspended in 1 mL wash buffer  pre-warmed to 37°C and incubated in a 37°C hybridization oven with rotation for another 30 min. Samples were washed for a total of four washes. After the last spin, samples are resuspended in 1 mL wash buffer and 150 μL removed for RNA isolation and the remaining 850 μL used for mass spectrometry.RNA isolation was performed using a modified version of a previously published protocol . RNA samCT value for three technical replicates of U6 for each sample. The normalized values were then used to calculate fold enrichment relative to Col-0 input.qPCR was performed as described above with the following exceptions. A standard curve for all primer sets was generated using serial dilutions of genomic DNA. “Copy number” of each transcript was calculated, and normalized by the average 4HCO3, and ultimately resuspended in 400 μL 100 mM NH4HCO3 supplemented with 200 mM NaCl and incubated overnight at 65°C to reverse crosslinks. The next day the samples were flash frozen in liquid nitrogen and stored at –80°C until processing. Samples were thawed on ice and resuspended in an appropriate volume of the resuspension buffer [50 mM SDS and 50 mM triethylammonium bicarbonate (TEAB) final concentrations] and reduced with final 10 mM DTT  for 30 min at 30 °C, followed by alkylation with final 50 mM iodoacetamide  for 30 min at 30 °C. The samples were processed using an S-Trap™ column according to the protocol recommended by the supplier : loaded onto the column and digested with trypsin  in 1:10 (w/w) enzyme/protein ratio for 1 h at 47 °C.Protein samples were centrifuged at 3000 RPM for 5 min, supernatant removed, and the beads were wash three times with 100 mM NHPeptides eluted from this column were vacuum-dried and resuspended with LC-MS grade water containing 0.1% (v/v) TFA for mass spectrometry analysis. Each sample was analyzed by a Q-Exactive HF mass spectrometer  coupled to a Dionex Ultimate 3000 UHPLC system  equipped with an in-house made 15 cm long fused silica capillary column (75 μm ID), packed with reversed−phase Repro−Sil Pur C18−AQ 2.4 μm resin  column. Elution was performed using a gradient from 5 to 35% B (50 min), followed by 90% B (10 min), and re-equilibration from 90 to 5% B (5 min) with a flow rate of 400 nL/min . Data were acquired in data-dependent MS/MS mode. Full scan MS settings were: mass range 200-1500 m/z, resolution 120,000; MS1 AGC target 1E6; MS1 Maximum IT 100. MS/MS settings were: resolution 30,000; AGC target 5E4; MS2 Maximum IT 200 ms; fragmentation was enforced by higher-energy collisional dissociation with stepped collision energy of 25, 27, 30; loop count top 15; isolation window 1.4; fixed first mass 120; MS2 Minimum AGC target 2E3; charge exclusion: unassigned, 1, 7, 8, and >8; peptide match preferred; exclude isotope on; dynamic exclusion 45 s.via Proteome Discoverer 2.4 with the default QExactive Precursor Quant and LFQ MPS with SequestHT and Percolator processing template and Comprehensive Enhanced Annotation LFQ and Precursor Quant consensus template with the following parameters. The spectra match with peptide sequence was performed with SequestHT with contaminants.fasta from MaxQuant and Arabidopsis thaliana fasta 2019.04 release, full tryptic digestion, maximum missed cleavage 3, peptide length between 6 and 144, MS1 mass tolerance 10 ppm, MS2 mass tolerance 0.02 Da, dynamic modification with oxidation on methionine, acetylation and methionine loss on protein N-terminal, static modification with carbamidomethyl on cysteine. The protein inference and identification validation was performed with Percolator with a 1% false discovery rate (FDR) cut off. Normalization was performed by total peptide amount and scaling mode was set to on all average. Protein Abundance was peptide summed with the top three most abundant peptides for each protein.The acquired data were processed lncCOBRA1 probes in Col-0 background were considered. Protein abundance in lncCOBRA1 pulldown was then normalized by the average protein abundance identified using the scrambled probes (lncCOBRA1/scrambled) and only proteins that were enriched with the lncCOBRA1 probes compared to the scrambled sequence probes were examined further (COBRA1/scrambled > 1). Enrichment of Col-0 over lnccobra1-2 background was then calculated as the log2[Col-0/lnccobra1-2] and proteins enriched over 1-fold were classified as lncCOBRA1-interacting and used for future analyses. We also examined proteins that were present in at least 2 biological replicates of the lncCOBRA1 pulldown in Col-0 tissue, but absent from scrambled and in 0 or 1 biological replicate of the lncCOBRA1 pulldown in the lnccobra1-2 background. Since no protein abundances were found in the scrambled, a fold enrichment could not be calculated.Proteins were first filtered such that only proteins with abundance scores in at least two biological replicates of the pulldown with the 5 was used to generate the protein-protein interaction network with medium stringency and clustered into five clusters by the k-means clustering algorithm provided. PPI enrichment was calculated by the STRING program (STRING program .Long non-coding RNAs (lncRNAs) are an important yet understudied class of molecules in all eukaryotic organisms. While thousands of lncRNAs have been identified, only a handful have described functions. In this work, the authors describe a previously uncharacterized and highly conserved lncRNA that contains two snoRNA domains, and functions to affect Arabidopsis germination and growth. Overall, the authors describe sno-lncRNAs for the first time in a non-mammalian system and demonstrate novel mechanisms of lncRNA function in Arabidopsis.PXD033707.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The ProteomeXchange Consortium via the PRIDE (1) partner repository with the dataset identifier MCK and BAG conceived the study and wrote the manuscript with assistance from all authors. MCK, HJK, KRP, and BAG designed the experiments. MCK, HJK, and KRP performed experiments and analyzed the data. All authors have read and approved the manuscript for publication.EL was employed by CyVerse Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here, we describe a small enterovirus outbreak including nine cases of aseptic meningitis in a New Zealand hospital in 2017. Most patients had a lymphocytic predominance in the CSF, their length of stay was short, and there were no paediatric cases or ICU admissions. VP1 genotyping revealed that the outbreak was caused by an echovirus E30 strain closely related to strains reported from the US, UK, Brazil, and Denmark. They all form a separate cluster within lineage “h”, which leads to the proposal of establishing a new lineage tentatively named “j” for this group of echovirus E30 strains. However, whole genome sequencing and reference mapping to echovirus E30 sequences showed very poor mapping of reads to the 3′ half of the genome. Further bioinformatic analysis indicated that the causative agent of this outbreak might be a mosaic triple-recombinant enterovirus composed of echovirus E6, echovirus E11, and echovirus E30 genome segments. Main drivers of enterovirus outbreaks in New Zealand were Coxsackie virus A24 (1997), Echovirus E33 (2000), Echoviruses E13 and E30 (2001/2002), Enterovirus type 68 (2010), and Coxsackie virus A6 (2013). In 2016 and 2017, the most predominant enterovirus serotypes were Coxsackie virus A6, Echovirus E18, and Echovirus E30 (22 laboratory-confirmed cases).Enteroviruses are viral pathogens causing a wide spectrum of diseases in humans ranging from mild common cold-like symptoms, hand foot and mouth diseases, acute haemorrhagic conjunctivitis, myocarditis, acute flaccid paralysis (including poliomyelitis), to fatal aseptic meningitis or encephalitis . NationaWe became aware of an unusual clustering of enteroviral meningitis cases caused by Echovirus E30 in Ashburton, New Zealand over several months in 2017. Ashburton is a small district with a population of about 32,400 people. Echovirus E30 is a member of the enterovirus genus and belongs to human enterovirus species B (HEV-B). It has been associated with large outbreaks of aseptic meningitis in adults and children in the community [Samples: Cerebrospinal fluid (CSF) samples from patients with aseptic meningitis were collected in Ashburton Hospital between August and November 2017.RNA extraction: Nucleic acids were extracted with the standard EasyMag  generic 2.0 extraction protocol .Neisseria meningitidis in pool 3.Real-time PCR: Real-time RT-PCR molecular diagnostic testing of the 9 CSF samples was completed with an in-house meningitis/encephalitis PCR panel targeting herpes simplex virus 1/2 plus varicella zoster virus in pool 1, enterovirus, and parechovirus in pool 2  and Neis2), 0.8 µL dNTPs (10 mM each), 1 µL 224 primer (50 µM), 1 µL 222 primer (50 µM), 0.5 µL FastStart Taq polymerase , and 31.7 µL water. The reaction was incubated for 10 min at 95 °C to activate the Taq polymerase. This was followed by 40 cycles of 95 °C for 30 s, 42 °C for 30 s, and 60 °C for 45 s. The programme finished with a final elongation at 72 °C for 5 min. Round 2 PCR was carried out in a 25 µL reaction volume containing 1 µL round 1 PCR product, 2.5 µL 10× Roche FastStart Taq Buffer (with MgCl2), 0.4 µL dNTPs (10 mM each), 0.4 µL AN89 primer (50 µM), 0.4 µL AN88 primer (50 µM), 0.25 µL FastStart Taq polymerase , and 20.05 µL water. The reaction was incubated for 10 min at 95 °C to activate the Taq polymerase. This was followed by 40 cycles of 95 °C for 30 s, 60 °C for 20 s, and 72 °C for 20 s. A final elongation at 72 °C was performed for 5 min. Round 2 PCR amplicons (384 bp region of the VP1 gene) were visualised by agarose gel electrophoresis. Sanger sequencing was performed using a BigDye Terminator v3.1 ready reaction cycle sequencing kit on an ABI Prism 3130xl automated sequencer  with primers AN232 and AN233 in separate reactions.Genotyping: VP1 gene genotyping was performed using a 2-step nested RT (reverse transcriptase)-PCR method described by Nix et al. , with moUnbiased whole genome sequencing: We were able to isolate sample 17WQ2027G collected on the 20 August 2017 on MRC-5 cells. Host cells were lysed by freeze–thawing, and host nucleic acids were depleted using benzonase  treatment for 2 h at 37 °C. The benzonase was inactivated by the addition of 0.5 M EDTA , and viral nucleic acids were extracted using EasyMag  generic 2.0 extraction protocol and quantified via a Qubit RNA HS Assay kit . First-strand cDNA synthesis was carried out for 1 h at 60 °C with Transcriptor Reverse Transcriptase  using random hexamer primers. The NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module  was used for second-strand cDNA synthesis at 16 °C for 1 h. Double-stranded cDNA was purified using AmpureXP beads  and quantified using Qubit High Sensitivity DNA assay . Barcoded NGS libraries were generated following the Nextera DNA Flex protocol . The sequencing was performed on MiSeq using Reagent Kit v2 (300 cycles) micro flow cell.Bioinformatic analysis: Maximum-likelihood phylogenetic analysis of the partial VP1 genotyping sequences and whole genome sequences was carried out using MEGA v5.1 after ClustalW alignment, which was inferred by bootstrapping for 500 replicates. De novo genome assembly was performed with SPAdes v3.11.0  and refeNine patients were hospitalised over a 13-week period spanning August to November in 2017. The age range was 16 to 55 years  and included a husband and wife. The median length of hospital stay was one day, and none required ICU admission. Presenting symptoms were headache (100%), photophobia (88.9%), fever (88.9%), neck stiffness (66.7%), and nausea and/or vomiting (77.8%). Out of all the patients, 7/9 had a CT head, of which all were normal bar one, which showed mild mucosal changes of the sinuses.All samples had raised white cell count (WCC) in the cerebrospinal fluid (CSF) , 8/9 had a lymphocytic predominance, CSF protein was 0.32–1.24 g/L, and CSF glucose was 3.1–3.9 mmol/L. Peripheral WBC was 4.4–13.0. The CRP range was 4–63 mg/L (for details, see All show the closest clustering to samples from “lineage h” as determined by comparison with strains described in the publication by Miyoshi et al. 2020 . HoweverA Simplot BootScan analysis  indicateThis could explain the poor mapping result of the 3′ half of the genome to other echovirus E30 genomes available in GenBank in 2017. However, the E30, E6, and E11 echoviruses used for the Simplot BootScan recombination analysis are not prototype strains and therefore could be recombinants in the 3′ half of the genomes themselves. An additional Simplot BootScan analysis of an alignment of the NZ Echovirus E30 MW586892 with available whole genome sequences of the enterovirus prototype strains described in  is preseThere is little current data about which enteroviruses are circulating in New Zealand. After a cluster of aseptic meningitis cases appeared in Ashburton, New Zealand, we decided to analyse the cases via VP1 genotyping. This revealed that the outbreak was caused by echovirus E30, with the closest clustering to strains from lineage h. However, since the reported echoviruses closely cluster with other echovirus E30 strains from 2014 and 2017 detected in the US, UK, Brazil, and Denmark, which form a separate distinguishable clade in the phylogenetic VP1 tree, we suggest grouping them into a newly established lineage tentatively named “j”. Echovirus E30 has been associated with outbreaks of aseptic meningitis globally and has the potential to cause very large outbreaks ,14. In kOur whole genome data analysis indicates that this newly discovered echovirus might be a mosaic triple-recombinant between echovirus E30, echovirus E6, and echovirus E11, which were most probably derived via intertypic/heterotypic homologous recombination during co-infection. However, the E6 and E11 strains that show the highest homology in the P2 and P3 regions could themselves be recombinant viruses as they were probably only classified based on their VP1 sequence. A comparison with enterovirus reference sequences showed no matches to E6 and E11 protype strains in the 3′ half of the genome see . TherefoIn addition, our results show that genotyping based on partial VP1 Sanger sequences might not be sufficient to characterise echovirus outbreaks, whereas whole genome sequence data provide much more detailed information, especially in regard to recombination events. Since the VP1 genotyping window is located in the 5′ half of the genome, it will always show the highest homology to a certain strain, such as in this case echovirus E30, even if the remainder of the genome has undergone recombination events with other echovirus strains or other enterovirus species. Interestingly, closely related sequences of this new triple-recombinant echovirus variant have also been detected in big aseptic meningitis outbreaks in California and Mongolia, implicating that this new variant has the potential to cause aseptic meningitis epidemics possibly due to new antigenic properties or altered replication or pathogenicity. Therefore, continuous molecular epidemiological surveillance via whole genome sequencing could provide a tool to better understand the true complexity and evolution of enterovirus genomes and their association with outbreaks and certain clinical manifestations.


Ascomycota phylum. Colletotrichum was the most dominant fungal genus accounting for 37% of all the isolates, followed by Diaporthe (13%), Phyllosticta (10.9%) and Diplodia (8.7%). Chemical elicitation using 5-azacytidine, a DNA methyltransferase inhibitor and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor resulted in an increase in the number of active strains. Bioassay-guided isolation and structural elucidation yielded pestahivin and two new analogues from Bartalinia sp. F9447. Pestahivin and its related analogues did not exhibit antibacterial activity against Staphylococcus aureus but displayed strong antifungal activities against Candida albicans and Aspergillus brasiliensis, with IC50 values ranging from 0.46 ± 0.06 to 144 ± 18 µM. Pestahivin and its two analogues furthermore exhibited cytotoxic activity against A549 and MIA PACA-2 cancer cell lines with IC50 values in the range of 0.65 ± 0.12 to 42 ± 5.2 µM. The finding from this study reinforces that chemical epigenetic induction is a promising approach for the discovery of bioactive fungal secondary metabolites encoded by cryptic gene clusters.The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of fungal endophytes obtained from the A*STAR Natural Organism Library (NOL) and previously isolated from Sungei Buloh Wetland Reserve, Singapore. Phylogenetic analysis based on ITS2 gene suggests that these isolates belong to 46 morphotypes and are affiliated to 23 different taxa in 17 genera of the  Ascomycota. Within the ascomycetes, classes Sordariomycetes and Dothideomycetes have been found to constitute the highest number of foliar fungal endophytes  using an initial denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, with a final extension step of 10 min of 72°C. Successful amplification of target region was confirmed by visualizing 1 µL of the products following Gel electrophoresis and the products were purified using the MEGAquick-spin™ Total Fragment DNA Purification Kit  before sending the amplicons to 1st BASE  for DNA sequencing. Using the software Benchling (https://benchling.com), the consensus sequences were constructed by alignment of the forward and reverse sequences. Sequence regions upstream of the forward primer and downstream of the reverse primer were removed from the aligned sequences and the resultant sequences uploaded into BLASTn (TEF-1α), RNA polymerase II second largest subunit (RPB2) and β-tubulin gene sequence (TUB) were obtained from F9447 genome data and those of closest relatives retrieved from NCBI database and recent references  following the manufacturer’s instructions. PCR amplification of the ITS2 region was performed using primer pair ITS4/ITS86F . The fino BLASTn  for strao BLASTn . All priferences . DNA genferences . The seqectively . For eacectively .The endophytic isolates were subjected to small-scale fermentation for extract generation by inoculating 3 mm fungal mycelial disc of 4–7 days old fungal culture into 10 ml pre-sterilized CF02LB and CF18LB media  in 50 mlStaphylococcus aureus strain ATCC 25923, a yeast Candida albicans strain ATCC 10231 and a mould Aspergillus brasiliensis strain ATCC 16404 were used to assess the antimicrobial activity of fungal secondary metabolites. Primary antimicrobial screening of fungal extracts was performed at concentration of 200 μg/ml in duplicate using the 384-well assay described previously  before the filtrate was dried using a rotary evaporator.Active extracts were analyzed according to a dereplication procedure as described in the literature . Strain 2O in a ratio of 1:1, 800 mL) and CH2Cl2 (400 mL), followed by extracting the aqueous methanol phase with CH2Cl2 (2 × 400 mL) (2Cl2 crude extract (13.8 g) was redissolved in CH2Cl2:MeOH (1:1) and separated by size exclusion chromatography on Sephadex LH-20 (CH2Cl2:MeOH = 1:1) and the sample (1.5 g) was subjected to silica gel column chromatography eluted using 1–5% MeOH/CH2Cl2 to obtain an enriched fraction containing pestahivin analogues (122.3 mg). The mixture of pestahivin analogues was further purified by C18 reversed-phase preparative HPLC  to give 37 mg of pestahivin (1) (RT 45 min), 20.5 mg of pestahivin B (2) (RT 34 min) and 7.3 mg of pestahivin C (3) (RT 39 min). The structure of pestahivin was confirmed by comparison of accurate mass from HRMS data and 1D/2D nuclear magnetic resonance (NMR) data with the literature values (The dried extract was combined and partitioned between aqueous methanol (MeOH:H 400 mL) . The orge values .2 spectra. The operating parameters for QTOF were the same as in previously reported  workflow using spectral clustering algorithm with a minimum matched fragment of 6 and a minimum pairs cosine of 0.7. The resulting molecular network was visualized and processed using Cytoscape version 3.9.0.Optical rotations were recorded using JASCO P-2000 digital polarimeter. NMR spectra were acquired on Bruker DRX-400 NMR spectrometer equipped with Cryoprobe. Preparative HPLC analysis were performed with Agilent 1,260 Infinity Preparative-Scale LC/MS Purification System and Agilent 6130B single quadrupole mass spectrometer using Agilent 5 Prep C18 column (10 × 30 mm). UV absorptions were measured, and HPLC-MS was done using Agilent UHPLC 1290 Infinity hyphenated with a diode array detector (DAD). HRMS spectra were recorded in positive ionization mode on an Agilent 6,540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer equipped an electrospray ionization (ESI) source. For over 8.6 min, a gradient condition of 98% water (0.1% formic acid) to 100% acetonitrile (0.1% formic acid) was employed, using an Acquity UPLC BEH C18  column, at flow rate of 0.5 ml/min. Collision energy of 40 eV was applied in the acquisition of MSreported . The acqvia PCR sequencing of the ITS2 region and LSU region for the accurate identification of species/genus level  and Botryosphaeriales (20%) followed by Diaporthales (13%). At the genus level, Colletotrichum was the most dominant grouping of fungi. Colletotrichum is an important plant pathogen but also occurs in other lifestyles such as endophytes, saprobes and rarely entomopathogens  housed at the Singapore Institute of Food and Biotechnology Innovation (SIFBI). A total of 46 isolates were successfully revived. In order to verify the taxonomic placement of the fungal isolates, gDNA was extracted and amplified us level . The Bla8 orders . Many phromycota , and theathogens . The genathogens . A previingapore . Besides Xylaria . Our finrequency . ITS/28Srequency . The detS. aureus, C. albicans and A. brasiliensis) and three cancer cell lines . .Chemical elicitation using small molecules to perturb the chromatin machinery has been identified as an important tool for the activation of cryptic secondary metabolite pathways in fungi . In the S. aureus, C. albicans and A. brasiliensis, respectively. Out of the 16 extracts showing antimicrobial activity, 9 (56%) and 7 (44%) were derived from fungal strains grown in CF18LB and CF02LB media, respectively. Regarding cytotoxic activity, 10 (7.3%), 7 (5.1%), and 1 (0.7%) revealed cytotoxic activity against A549, MIA PaCa-2 and PANC-1 cell lines, respectively, with 8 (73%) and 3 (27%) of the 11 active extracts arising from fungal strains grown in CF18LB and CF02LB media, respectively. Differences in the bioactivity of fungal extracts derived from the same strains when grown in different media have been reported in the literature  exhibited antimicrobial and/or cytotoxic activity against at least one of the tested microbial pathogens and cancer cell lines. terature . Extractterature . Thus, tectively .http://dnp.chemnetbase.com). This resulted in the putative identification of three known bioactive molecules from 2 strains (m/z 309.2298) and fumitremorgin C (m/z 380.1967) identified in F6430 fermented in CF18LB media in the presence of 50 µM 5-azacytidine or 100 µM SAHA demonstrated antifungal activities while new pestahivin analogues  and pestahivin (m/z 977.6425) identified in F9447 fermented in CF02LB in the presence of 50 µM 5-azacytidine or 100 µM SAHA presented both antifungal and cytotoxic activities. Unknown antibacterial compounds  were identified in F6430 fermented in CF18LB media in the presence of 100 µM SAHA  (m/z 977.6425) in F9447 by ∼3 -fold, and pestahivin B (2) (m/z 935.5953) and pestahivin C (3) (m/z 949.6113) by 3-4-fold in F9447  and the two new pestahivin analogues, pestahivin B (2) and pestahivin C (3) that were responsible for the observed antimicrobial activities  and 7 amide/carbonyl carbon signals . In addition, the 1H/13C NMR, HRMS, and specific rotation data of compound 1 were consistent with the literature values   .2 was isolated as white amorphous powders and assigned the molecular formula C50H78N8O9 following (+)-HRESIMS data analysis  of LEU-7 to carbonyl carbon (δC 167.7) of MTO-6 while the connection between MALA-1 and LEU-7 was deduced from the HMBC correlation between the N-methyl protons (δH 3.18) of MALA-1 and carbonyl carbon (δC 170.3) of LEU-7 (2 has been previously synthesized by 1H NMR data of compound 2 (2 was established and given the name pestahivin B (2). Notably, this is the first report of isolation of 2 from a natural source.Compound analysis . Comparitra of 2 . The conof LEU-7 . Literatmpound 2  were con3 was isolated as yellow amorphous powders. (+)-HRESIMS measurement established the molecular formula C51H80N8O9. Comparison of NMR and MS data between 1 and 3 suggested that the latter natural product was missing a PrLEU residue, and this residue was replaced with an unnatural amino acid residue, MeLEU (3 as supported by the HMBC correlation from the amide proton at δH 8.62 of MeLEU-7 to carbonyl carbon (δC 167.9) of MTO-6 in addition to the HMBC correlation between the N-methyl protons (δH 3.19) of MALA-1 and carbonyl carbon (δC 170.5) of MeLEU-7   of 1 continued to lose a unit of LEU (113.0841 Da). The sequential loss of a unit of Leu explained the occurrence of fragment ion m/z 833. 5,394 . This is followed by the sequential loss of a unit of MLEU with 155.0946 Da that explained the occurrence of fragment ion m/z 677.4390 . The continued loss of a unit of PrLEU-DGCN with 222.1732 Da led to the formation of a fragment ion m/z 458.2845 . The subsequent loss of a unit of MALA with 130.0504 Da and a unit of PrLEU with 155.1310 Da explained the occurrence of the final fragment ion of MTPO m/z 173.1062 . This fragment ion resulted from a neutral loss of a C=O. In total, the fragmentation pathway of compounds 1–3 took four major steps of losing a unit of LEU, a unit of MLEU, a unit of PrLEU-DGCN and a unit of MALA. However, since compounds 2 and 3 has different substitution at the seventh residue, the last fragmentation step is the losing of a unit of LEU with 113.0841 Da in compound 2 and a unit of MeLEU with 127.0997 Da in compound 3 (1–3.To further confirm the structures of compounds and MTO) . After tmpound 3 . In addimpound 3  further 1) and pestahivin B (2) and C (3) are provided in The NMR spectra of pestahivins , see 1H and 13C NMR data, see White amorphous powders; max 224 nm; HR-ESI-MS m/z 935.5976 [M+H]+ , see 1H and 13C NMR data, see White amorphous powders; max 224 nm; HR-ESI-MS m/z 949.6120 [M+H]+ , see 1H and 13C NMR data, see Yellow amorphous powders; 1) and its new analogues pestahivins B (2) and C (3) isolated from Bartalinia sp. F9447 were subjected to antimicrobial activity evaluation against S. aureus, C. albicans and A. brasiliensis and cytotoxicity testing against A549, MIA PaCa-2 and PANC-1 cell lines. Pestahivin (1) and its analogue C (3) showed similar antifungal activity profiles against C. albicans with IC50 value of 1.9 ± 0.26 µM, and similar activity against A. brasiliensis with IC50 values of 0.46 ± 0.06 and 0.65 ± 0.07 µM, respectively (3) showed 45-fold and 7-fold more potent IC50 against C. albicans and A. brasiliensis respectively, when compared to pestahivin B (2). No antibacterial activity against S. aureus was observed for all three compounds. All three compounds demonstrated more potent cytotoxic activity against MIA PaCa-2 than A549 cells. There was no significant inhibition against PANC-1 pancreatic cell line, where the inhibitory activity plateaued at 20–30% inhibition. The dose-response curves for the three compounds are shown in Pestalotiopsis sp. RF-5890 strain and was found to exhibit a strong antiviral effect against the human immunodeficiency virus (HIV)  . Interesβ-tubulin sequence dataset ; however, strain F9447 is significantly different from B. bella CBS 464.61, B. pini CBS 143891 in RPB2 region (18 and 54 nucleotide bases), TEF region (35 and 84 nucleotide bases), and β-tubulin region (19 and 52 nucleotide bases). Similarly, a comparison of ITS region shows that previously published reference strain B. pini CBS 143891 is not significantly different from B. robillardoides CBS 122615 (only 2 differentiated nucleotide bases); however, B. pini CBS 143891 is different from B. robillardoides CBS 122615 in 43, 81, and 58 nucleotide bases in RPB2, TEF and β-tubulin regions respectively. Strain F9447 is therefore a potentially new species based on LSU, ITS, TEF, RPB2 and β-tubulin sequence dataset. We therefore conclude that strain F9447 is possibly a new species in Bartalinia genus which was isolated from grass flower collected from Sungei Buloh Wetland Reserve, Singapore. This strain was found to produce new pestahivin analogues whose biosynthesis was enhanced in the presence of two chemical elicitors.Phylogenetic analyses of a combined LSU, ITS, TEF, RPB2 and  dataset  show thaAscomycota with Colletotrichum being the most dominant fungal genus. Extracts generated from 23 fungal strains grown in two media with or without two epigenetic modifiers exhibited varied levels of antimicrobial and cytotoxic activities. One fungal strain, Bartalinia sp. F9447 exhibited enhanced antifungal activity when grown in the presence of 5-azacytidine. Large-scale fermentation and isolation studies led to the isolation of pestahivin and its two novel analogues. The levels of production of the three pestahivin compounds were greatly enhanced when the fungal strain was grown in the presence of two chemical elicitors. To the best of our knowledge, this is the second time that pestahivin has been isolated from fungi, and the first time that two of its analogues are being reported. The study thus further demonstrates that chemical elicitation using epigenetic modifiers is an important tool for enhanced production of constitutive fungal secondary metabolites or for discovery of new natural products.In this study, phylogeny, antimicrobial and cytotoxic potential of endophytic fungi isolated from Sungei Buloh Wetland Reserve, Singapore were investigated. Our findings provide an insight into the species richness of endophytic fungal community from mangrove and other plants growing in this area. Overall, our results reveal the presence of a highly diverse endophytic fungal community in the study area which may include potential novel undescribed strains. All the studied fungal strains were affiliated to the phylum