Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation.RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the  RNA viruses include the most abundant group of human, animal and insect pathogens. These viruses encode a specialized enzyme, the RNA-dependent RNA polymerase (RdRP), responsible for RNA genome replication and transcription. All known RdRPs share a similar closed right-hand architecture, with the fingers, palm and thumb subdomains and use the same mechanism of catalysis . This haRdRPs function either as single polypeptides or in a complex with other viral or host components to transcribe and replicate the viral RNA genome. They use a variety of strategies to recognize substrates and coordinate the different steps of RNA synthesis .2+ ions that are crucial for catalysis and pyrophosphate release triphosphate) were obtained from Jena bioscience.pol-Δ10). Vector modification consisted of the addition of the coding sequence for the Tobacco etch virus (TEV) protease site downstream of the sequence coding for the His tag, in a BamHI site. The resulting recombinant protein contained a removable hexa-histidine tag at the protein N-terminus.cDNA from TaV ORF1 was obtained from Genescript as previously described . The DNAE. coli (DE3). The presence of the designed deletion in the final plasmid (pACYCDuet-Δ10-TaVpol-Δ607-624) was confirmed by DNA sequencing.Deletion in the priming loop (amino acids 607-624) was performed by PCR in a 50 µL reaction, using a site-directed mutagenesis protocol previously described , in whicpol versions, Δ10-TaVpol and Δ10-TaVpol-Δ607-624, were transformed into the E. coli BL21 (DE3) strain and cultured at 37 °C in LB media containing 35 µg/mL of chloramphenicol until OD600 = 0.7, induced with 0.1 mM isopropyl-ß-D-1-thiogalactopyranoside (IPTG) and incubated ON at 22 °C. Cells were harvested by centrifugation  and the bacterial pellet was resuspended in lysis buffer or buffer A  supplemented with a protease inhibitor cocktail , 10 µg/mL of DNAse , 10 µg/mL of RNAse (Neo-Biotech), 0.1 mg/mL of lysozyme and 5 mM MgCl2. Cells were homogenized at 4 °C on a cell disrupter at 1.4 MPa  and the extract clarified by centrifugation at 48,000g for 30 min in a JA25.50 rotor (Beckman Coulter) at 4 °C. The supernatant was filtered  and loaded onto a buffer A pre-equilibrated 5 mL His-trap HP column  with 0.5 mL/min flux. The column was washed with 10 column volumes (CV) of lysis buffer without additives, mixed with 20% of buffer B  and eluted with a linear gradient of 30 CV until 100% B.The recombinant plasmids to produce the TaVv/v glycerol and 2 mM DTT. The dialyzed protein was subsequently treated with TEV protease (0.02 mg/mL) ON at 4 °C, then incubated with TALON resin (Takara) for 1 h at 4 °C and centrifuged to eliminate TEV. The supernatant was further purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column , exchanging the buffer to 50 mM MES pH6, 250 mM NaCl, 5 mM DTT and 5% v/v glycerol. Finally, fractions containing the purified protein were pooled and concentrated using Amicon Ultra 10K filters (Millipore) to get 40 mg/mL.Fractions containing the protein of interest were pooled and dialyzed ON at 4 °C against buffer 50 mM Tris pH 7.5, 300 mM NaCl, 5% pol and Δ10-TaVpol-Δ607-624 were performed by the sitting-drop vapor diffusion method at 293 K with a 200 nL drop (1:1 ratio) in 96-well Greiner plates, using a nanoliter-drop dispenser robot  and several screens  available at the automated crystallography platform (PAC) . Crystals appeared after 1 day, in different conditions. Several rounds of optimization conditions (protein and precipitant concentrations and drop size) were performed to obtain suitable crystals for X-ray studies. The best Δ10-TaVpol crystals were obtained in 19% PEG 3350, 0.18 M MgSO4. The Δ10-TaVpol-Δ607-624 construct (unbound-form) was crystallized in 0.1 M sodium acetate pH 4.6, 0.25 M (NH4)SO4 and 26% PEG4000. The Δ10-TaVpol-Δ607-624-RNA complex was obtained and crystallized as follows: The partially autocomplementary RNA oligonucleotide 5′CAAAAUUU3′ was resuspended in water (previously treated with 0.1% v/v DEPC) at a 4 mM concentration and annealed by heating at 90 °C during 1 min and slowly cooling to 10 °C at a rate of 1 °C/min on a PCR machine. The dsRNA molecule was then added to the Δ10-TaVpol-Δ607-624 protein solution in an equimolar proportion. The nucleotides UTP or a mix of UTP and GTP were also added at 2 mM concentration and the solution was incubated, for 2 h at 4 °C before starting the crystallization trials. Well diffracting crystals of the TaVpol-RNA complex were obtained in two days from a solution, containing 0.1 M sodium acetate pH 4.6, 0.22 M (NH4)SO4 and 28% PEG2000MME. These crystals were soaked with 2 mM MgCl2 for a few minutes in the cryobuffer solution  glycerol) to obtain the ternary Δ10-TaVpol-Δ607-624-RNA-UTP and Δ10-TaVpol-Δ607-624-RNA–uridine-5’-[-imido]triphosphate complexes.The initial crystallization trials of Δ10-TaVAll crystal optimization experiments were performed at 293 K, by sitting drop vapor diffusion, in an MRC 48 well plate using a 200 µL reservoir of the crystallization buffer and a 1 µL drop (1:1 ratio).v/v) glycerol and flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K on the BL13-XALOC beamline at the ALBA Synchrotron  and the P13 and P14 beamlines, operated by EMBL at the PETRA III storage ring .Crystals were harvested in cryo-loops (Molecular Dimensions), soaked for 1 min in a reservoir solution and 20% (pol apo-form (PDB: 4XHI) as a search model. Refinement was done alternating cycles of manual rebuilding using Coot triphosphate) and (iii) bound to a template/primer RNA, in the presence of a UTP/GTP mixture , able to form a 6-base pair duplex, flanked by two 2-nucleotide 5′ overhangs, acting as both an RNA template and primer. A self-complementary RNA plus two ssRNA nucleotides of equal sequence was previously employed to trap different elongation complexes of the HCV NS5B RdRP . The finpolΔ607-624 protein bound to RNA was very similar to the unbound TaVpol (root mean square deviation (r.m.s.d) values of 0.5 Å for the superposition of 540 residues), showing that no important rearrangements of the TaVpol conformation were induced by the RNA binding. Noticeable conformational changes, consisting of a main-chain movement of motif A towards motif C and the side chain repositioning of the catalytic D369 (motif A) near the active site, have been observed in all RNA complexes -imido]triphosphate were obtained to further characterize different steps in the process of RNA synthesis  but far away of the triphosphate moiety of the rNTP analog that was salt bridged with the basic side chains K488 (motif D) and R280 of motif F in the rNTP tunnel , Asp351 (Motif C) and the pyrophosphate (PPi) byproduct. The PPi molecule was also salt bridged by K488 (motif D) and R280 (motif F) in the rNTP tunnel , revealed the existence of a second metal ion-binding site located nearby the catalytic cavity, in a non-catalytic position. The resulting electron density maps at 2.07 Å resolution showed strong peaks of extra density in the two molecules of the asymmetric unit. The Mg2+ ions were located at 7 Å from the catalytic aspartate D351 (motif C), coordinated by the side chain carboxyl oxygen of D352 (motif C) and five water molecules, in a perfect octahedral geometry , P368 (O)M w4, with the main chain oxygen of K51: w3 and with the main chain oxygen of C3676: w5  shows a 25° hinge movement with respect to the same position in the RNA-bound Δ10-TaVpolΔ607-624  is structurally equivalent to the priming loops of flaviviruses and bacteriophage ϕ6 triphosphate (2.4 Å resolution) by cocrystallization, followed by soaking with Mg2+ (the UTP complex) or Mn2+ -imido]triphosphate complex) ions into ternary form cocrystals.The TaVphage ϕ6 ,23,24,28e chains . Biochemphage ϕ6 ,12,29. Hpol and Δ10TaVpolΔ607-624 complexed with the template-primer oligonucleotide showed that no other large rearrangements, additional to λ6 loop elimination, were induced in the polymerase by the RNA binding. A noticeable local change included a main-chain movement of motif A, approaching motif C and the side chain repositioning of the catalytic aspartate of motif A (D369) towards the central cavity, closing the catalytic site ssRNA viruses have provided great insights into the RdRP rearrangements associated to the different steps of the catalytic cycle ,34,35,36pol active site occurred without completing the three-stranded β-sheet formed by motifs A and C  and HCV (D318–D220 5.7 Å)  d–f.pol compared to those of enteroviruses is that the active site closure takes place on RNA template-primer binding, before rNTP binding and is maintained during rNTP recognition  and D369 (motif A) still remained coordinated with the two Mg2+ ions. The position B Mg2+ in turn coordinated the ppi byproduct in the rNTP channel. The conserved residues R280 (motif F) and K488 (motif D) were bound the ppi molecule, opposite to metal ions.Another difference between the elongation complexes of TaVognition c,d but aognition d. The inpolΔ607-624-RNA-rNTP ternary complex was obtained in the presence of the nucleotide analog uridine-5’-[-imido]triphosphate to catch the TaV enzyme in a distinct catalytically relevant conformation (2+ A), leaving the metal B position empty  and canonical RdRPs.Altogether, the structural data presented here define the requirements for processive RNA synthesis by TaV


Serum samples up to 91 days were collected and the plasma concentrations of triptorelin and testosterone were analyzed with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In vivo pharmacokinetics showed that injections of the silica-triptorelin acetate depot gave 5-fold lower Cmax values than the corresponding Pamorelin® injections. The depot also showed a comparable sustained triptorelin release and equivalent pharmacodynamic effect as the Pamorelin® injections. Detectable triptorelin plasma concentrations were seen with the depot after the 91-day study period and testosterone plasma concentrations remained below the human castration limit for the same period.Triptorelin acetate was encapsulated into silica microparticles by spray-drying a mixture of colloidal silica sol and triptorelin acetate solution. The resulting microparticles were then combined with another silica sol containing silica nanoparticles, which together formed an injectable silica-triptorelin acetate depot. The particle size and surface morphology of the silica-triptorelin acetate microparticles were characterized together with the in vitro release of triptorelin, injectability and rheology of the final injectable silica-triptorelin acetate depot. In vivo pharmacokinetics and pharmacodynamics of the silica-triptorelin acetate depot and Pamorelin Silica is a well-known material in the field of controlled drug delivery. It has been used as a matrix material to delivery different types of active pharmaceutical ingredients (API) and silica has been manufactured into various dosage forms  . DependiThe other common approach for preparing silica for controlled drug delivery is the direct encapsulation/embedment of API during sol-gel processing, which involves incorporating an API into a colloidal silica nanoparticle solution . In practice, with the silica sol-gel processing method, there is no upper limit for the size of API that can be encapsulated/embedded into the nanoscale silica matrix. When the encapsulation/embedment into the silica matrix is effective, the API release is mainly based on matrix erosion . This controlled release driven by matrix erosion can be achieved when the resulting pore structure is smaller than the API or the pore size is so small that the diffusion rate of the API is neglectable in comparison to the dissolution rate of the matrix. The dissolution rate and subsequent release of the API is mainly controlled by adjusting and controlling the chemical structure of the silica matrix  ,5,6. TheDevelopment of minimally invasive drug delivery devices has also been one of the general aims in the field of drug delivery for quite some time. Recently the development has been accelerated by the new biological drugs for intraocular release , which h®, a PLGA-based formulation containing triptorelin pamoate, administered with a 20 G needle (outer diameter 0.908 mm). Injectable suspensions and hydrogels are often based on organic polymers, but there are also inorganic formulations available . Injectable dosage forms are often based on different types of microparticles in suspensions ,11, or oThe direct encapsulation/embedment of API in silica has been used for several different dosage forms, but a minimally invasive injectable depot formulation has been developed . Said si® was evaluated alongside the silica-based depot after subcutaneous injections in Sprague-Dawley male rats. The in vivo release of both formulations was investigated by conducting a pharmacokinetic study including the analysis of plasma testosterone levels to determine the pharmacodynamic effect of the released triptorelin.The aim of the study was to adapt the silica-based composite, comprising of silica microparticles embedded in a silica hydrogel, for a controlled and sustained delivery of triptorelin acetate (a decapeptide). Triptorelin acetate was chosen to show the potential of the silica technology for controlled delivery of peptides due to good pharmacodynamic and pharmacokinetic modeling. Hereafter, referred to as silica-triptorelin acetate depot. This included optimizing the injectability of the silica-triptorelin acetate depot, so that it could be administered using 22–25 G needles ensuring a minimally invasive delivery of the API. The silica-based depot was characterized both in vitro and in vivo. The in vitro studies include the characterization of the microparticles embedded into the hydrogel (particle size distribution and SEM-imaging). The rheological properties and injectability of the final silica-based depot were characterized. The controlled and sustained delivery of triptorelin acetate was analyzed both in vitro and in vivo. In vitro release was characterized by conducting dissolution experiments at in sink conditions for silica dissolution and triptorelin acetate. In the vivo study, commercially available Pamorelin®) and 0.1 M sodium hydroxide  were purchased from VWR Chemicals . Triptorelin acetate was supplied by Ferring Pharmaceuticals and Pamorelin® 11.25 mg, batch N19449 , was purchased. Testosterone standard for bioanalysis was from TCI  and internal standards alarelin acetate  and deuterated testosterone  were purchased from Sigma-Aldrich. All other materials and reagents used were of analytical grade and purchased from Sigma-Aldrich/VWR unless otherwise specified. Reagent grade tetraethyl orthosilicate (TEOS) was purchased from Sigma-Aldrich  and analytical grade solutions of 0.1 M hydrochloric acid  mixed with an alkaline triptorelin acetate aqueous solution. The alkaline triptorelin acetate solution was prepared by dissolving triptorelin acetate in deionized water and adjusting the pH of the solution to 10.1 with a 0.1 M NaOH solution. The final triptorelin acetate concentration in the solution was 0.7 mg/mL. The second component, the silica sol, was produced by hydrolysis of TEOS in deionized water using 0.1 M HCl as a catalyst. The molar water:TEOS:HCl ratio in the sol was 4:1:0.03. The hydrolysis reaction was carried out under strong mixing at room temperature and after the hydrolysis reaction the resulting silica sol was cooled down in an ice water bath. The spray-drying process was performed as a continuous feed process where the triptorelin acetate solution and the cold silica sol was fed into a tube reactor using a peristaltic pump and silicon tubing. The tube reactor was connected to the spray-dryer and the pumping rates of the two solutions were adjusted in way that the pH of the combined solution in the reaction tube was 4.6 and the triptorelin acetate solution feed rate was ten-fold compared to the silica sol feed rate. The spray-dryer was a Buchi B-290  and the aspirator air flow rate was 100%, atomization air flow was 670 L/h, total feed rate was 5.6 mL/min, and the inlet and outlet temperatures were 120 °C and 73 °C, respectively. w/v). The homogenous microparticle-sol suspension was then filled into disposable 1-mL BD Luer-Lok syringes, Catalogue no. 309628,  and stabilized into semi-solid gel within the syringes at room temperature for three days using a custom-made tube rotator . Noteworthy is that without the nanoparticles (derived from the R400 silica sol) the aqueous microparticle suspension is unstable at rest resulting in poor injectability. Just by incorporating ca. 0.5 wt.% silica nanoparticles is enough to transform the unstable microparticle suspension into a stable semi-solid injectable gel. To produce the injectable silica-triptorelin acetate depot, the silica-triptorelin acetate microparticles were incorporated in a R400 silica sol  and the resulting microparticle-sol suspension was stabilized into a semi-solid gel at room temperature for three days. The R400 silica sol was produced by hydrolysis of TEOS in deionized water, using a 0.1 M HCl solution as a catalyst, under strong mixing at room temperature. After the hydrolysis reaction, the pH of the R400 silica sol was set to 5.9 with 0.1 M NaOH solution. Then the silica-triptorelin acetate microparticles were suspended in the R400 silica sol with a 0.75:1 ratio  by analyzing the molybdenum blue complex absorbance at λ = 820 nm . v/v) and the mobile phase B was acetonitrile and formic acid in a ratio of 1000:1 (v/v). The injection volume was 10 µL, the column temperature was 30 °C and the flow rate was 1.0 mL/min. Mobile phase B (%) was kept constant at 8% for 5.5 min and then gradually increased to 90% over 1.5 min. After 1-min washout period, mobile phase B (%) was reduced back to 8%. Control and calibration samples were prepared with the triptorelin dissolution test buffer. In vitro degradation of silica and resulting release of triptorelin from the silica-triptorelin acetate depot was measured in 50 mM TRIS buffer pH 7.4 containing 0.01 % (v/v) TWEEN® 80 at 37 °C. The silica concentration in the dissolution buffer was kept at in sink conditions (free dissolution of the silica matrix) by dissolving 10–30 mg samples in 50 mL of the dissolution buffer. The dissolution buffer was changed to fresh medium at every sampling time point to keep silica concentrations below 30 ppm (at in sink condition). The dissolution studies were conducted for 7 days in a shaking water bath (60 strokes/min) at 37 °C . The dissolved silica was measured as described earlier. The released triptorelin was quantified by HPLC (Agilent Technologies 1260 Infinity) with a Waters XBridge C18 2.5 µm, 3.0 × 20 mm column. Herein, the mobile A phase consisted of water and trifluoroacetic acid in a ratio of 1000:2.5 (v/v) and the mobile phase B consisted of acetonitrile and trifluoroacetic acid in a ratio of 1000:2.5 (v/v). The absorbance was detected at 220 nm, the injection volume was 40 µL, the flow rate was 1.0 mL/min and the column temperature was 80 °C. Mobile phase B (%) was first gradually increased from 15% to 50% over 1.5 min followed by another increase from 50% to 99% over 1 min. After 0.5 min washout period, mobile phase (%) was reduced to 15%.The total triptorelin content was measured indirectly with a peptide hydrolysis method by dissolving silica-triptorelin acetate microparticle and silica-triptorelin acetate depot samples in strong alkali, simultaneously hydrolyzing triptorelin into amino acids. The hydrolysis was done in 3 M NaOH solution containing 5% 2,2′-thiodiethanol and 5 mg/mL amino acid mixture  at 100 °C for 24 h after which the tryptophan was quantified with a 1260 Infinity HPLC  connected to a multiple wavelength detector (at λ = 280 nm). The chromatographic separation was obtained on a Waters Symmetry C18 3.5 µm, 4.6 × 150 mm HPLC column. The mobile phase A was water and formic acid in a ratio of 1000:1 (® Rats) 9 weeks old and 250–300 g in weight, were subcutaneously administered 5 mg/kg or 10 mg/kg of triptorelin acetate. Triptorelin and testosterone plasma concentrations of four groups  of 6 animals (n = 6) after administration of Pamorelin® and the silica-triptorelin acetate depot were compared. The animal study was conducted by qualified animal technicians in the Central Animal Laboratory of Turku University (Finland) in compliance with the guidelines of the National Laboratory Animal Board of Finland. Specific Pathogen Free (SPF) Sprague-Dawley male rats  was prepared according to the package leaflet instructions: one vial of Pamorelin powder , mannitol, carmellose, sodium, polysorbate 80) was reconstituted in 2 mL of solvent (water for injection) provided in two ampules. The solvent was added to the powder using a syringe and injection needle provided in the package. Once the powder was reconstituted the resulting suspension was drawn into the syringe and the needle was changed to a sterile injection needle, also provided in the packaging, before administration. Pamorelin® was administered using a 20 G × 1” needle , whereas the silica-triptorelin acetate depot was administered using a 23 G × 1” needle . On the day of administration, Pamorelin®. Group II received 1.27 mg of triptorelin acetate in 100 µL injection of the silica-triptorelin acetate depot. Group III received 2.54 mg of triptorelin acetate in 200 µL injection of the silica-triptorelin acetate depot. Group IV received 2.26 mg of triptorelin acetate in 400 µL injection of Pamorelin®. Before and after dosing, blood samples were collected for 91 days and the plasma, from the blood samples, was frozen and stored at −20 °C for later analysis. Group I received 1.13 mg of triptorelin acetate in 200 µL injection of PamorelinThe triptorelin and testosterone concentrations of the plasma samples were simultaneously analyzed with UPLC-MS/MS . Sample preparation included solid-phase extraction (SPE) using HLB SPE plate . 40 µL of rat plasma sample was mixed with 400 µL of ultrapure water and 40 µL internal standard solution containing 20 ng/mL of alarelin acetate and testosterone-D3  and mixed. The SPE plate was first equilibrated by passing first 1 mL of methanol, and then 1 mL of water through each well. The samples were transferred onto the HLB plate and aspirated in the plate by applying 15 inHg vacuum for 10 min. The wells were then washed with 1 mL (60:40 Methanol-water) solution, followed by elution of the compounds with 1 mL of methanol containing 0.01% formic acid. The samples were transferred onto the HLB plate aspirated in the plate by applying 15 inHg vacuum for 10 min and evaporated to dryness in nitrogen flow and reconstituted into (60:40) methanol-water containing 0.01% formic acid. v/v) and the mobile phase B was acetonitrile and formic acid in a ratio of 1000:1 (v/v). The injection volume was 4 µL, the column temperature was 40 °C and the flow rate was 0.5 mL/min. Mobile phase B (%) was kept constant at 2% for 0.5 min and then gradually increased to 95% over 2.0 min. After 0.5-min washout period, mobile phase B (%) was reduced back to 2%. Capillary voltage used in the measurement was 1.0 kV (ESI+). The internal standards for testosterone and triptorelin were testosterone-D3 and alarelin, respectively. Quantitation of triptorelin was performed by acquiring MRM 656 (IS:584) > 249 (CE 26) in positive polarity with a cone voltage of 30 V. Testosterone was quantitated by acquiring MRM 289 (IS: 292) > 97.2 (CE 22). Cone voltage was 30 V. The chromatographic separation was obtained on a Waters Acquity BEH C18  column using Acquity UPLC connected to Xevo TQ-S triple quadrupole mass spectrometer  from Waters. The mobile phase A was water and formic acid in a ratio of 1000:1  using models Plasma Data, Extravascular Administration and Sparse Sampling. Calculation method used was Linear Trapezoidal with Linear Interpolation. The standard samples were prepared into female rat plasma by spiking the matrix into concentrations 0.1–5000 ng/mL of the analytes and otherwise treated as the samples. The quality control samples were prepared and treated identically with concentrations of 3, 30, 300, and 3000 ng/mL. To increase qualified sensitivity from previous prequalification, six Lower limit of quantitation (LLOQ) level quality control (QC) samples at 0.2 ng/mL for testosterone, and at 0.5 ng/mL for triptorelin, were added into the first samples run to prove the increased sensitivity of the method. Calibration curves were generated by quadratic fitting using weighting 1/x, resulting in R® were excised from the rats, frozen and stored at −20 °C before being analyzed for their triptorelin content. The silica-triptorelin acetate depot remnants were dissolved in 50 mL of 50 mM glycine buffer solution (pH 9.4), whereas the Pamorelin® remnants were immersed in 50 mL of ethanol. The dissolutions were carried out at 37 °C in a shaking water bath (60 strokes/min). The glycine buffer was periodically refreshed during sampling to maintain the free dissolution of the silica matrix (in sink conditions). The triptorelin concentration of the samples was measured with the HPLC method described earlier. The dissolution test was conducted until the release of triptorelin was not detectable by the HPLC method described previously. After necropsy on day 91, the remnants of the silica-triptorelin acetate depots and PamorelinParticle size distribution (PSD) and surface morphology of the silica-triptorelin acetate microparticles were characterized by laser diffraction and scanning electron microscopy (SEM), respectively. For the PSD measurements a HELOS 2370  instrument was used and for the SEM imaging, a LEO Gemini 1530 SEM  with a Thermo Scientific UltraDry silicon drift detector (SDD) was used. −1 to 1500 s−1 within 30 s, then decreasing the shear rate back to 1 s−1 within 60 s. The measuring gap was 0.3 mm. The viscoelastic properties  were studied with oscillatory measurements within the linear viscoelastic range of the samples with a measuring gap of 1.0 mm. The samples were studied under controlled deformation (γ < 0.002) within a frequency range of 0.1–10 Hz. All rheological measurements were performed at 25 °C.Rheological properties, such as viscoelasticity and thixotropy, of the silica-triptorelin acetate depot were characterized with a Haake RheoStress 300 rheometer  using a parallel plate (d = 20 mm) measuring geometry. The dynamic viscosity and thixotropic behavior were measured by linearly increasing the shear rate from 1 sThe injectability was estimated manually using a 25 G × 1” hypodermic needle. The needle was attached to the prefilled syringe and then the syringe was emptied. The injected volume was 400 µL and the injectability was evaluated as pass or fail. The sample passed if the prefilled syringe could be emptied with a single steady continuous push of the plunger.® 06 Plus kit . The measured endotoxin units (EU) were <0.06 EU/mg, which is below the Ph.Eur. limit of <0.3 EU/mg.The endotoxin content of the silica-triptorelin acetate depot was analyzed in accordance with EP 2.6.14. Method B , using Pyrogenw/w). Particle size distribution and SEM images of the silica-triptorelin microparticles, used in the manufacturing of the silica-triptorelin acetate depot, are shown in The triptorelin payload in the silica-triptorelin microparticles was 3%  were 1.30 µm ± 0.01 µm, 4.45 µm ± 0.01 µm and 17.12 µm ± 0.30 µm, respectively. The D-values indicate the portion of the particles in the sample are smaller than the given values . The SEM images show that the silica-triptorelin microparticle bulk consists of smooth spherical microparticles, dimpled spherical microparticles and broken shard-like microparticle shells. The bimodal density distribution measured for the microparticles can be explained by the presence of these different morphologies. The dimpled microparticles and the broken particle shells are smaller in size compared to the smooth spherical microparticles .The sol-gel parameters as well as the spray-drying conditions are known to impact the final morphology of the microparticles formed by spray-drying ,18,19. TThe silica-triptorelin acetate depot was manufactured by mixing spray-dried silica-triptorelin microparticles with a silica sol, which mostly consist of water (ca. 97 wt%) and low concentrations of solid silica nanoparticles (ca. 0.9 wt%). The high-water content silica sol will gradually gel on its own into a hydrogel, but together with the microparticles a stronger physical gel structure is formed. Fundamentally, the silica-triptorelin acetate depot is a particle-reinforced composite material that requires a certain amount of silica nanoparticles in the hydrogel as well as a certain amount of silica microparticles, embedded into the hydrogel, to maintain structural stability of the final depot . The phy®. The silica-triptorelin acetate depot was shown to be injectable through a 25 G needle, whereas Pamorelin® is administered using a 20 G needle. For the pharmacokinetic study, the silica-triptorelin acetate depot was injected using a 23 G needle to ensure low force of injection into the subcutaneous space of the rat.The gel structure of the semisolid silica-triptorelin acetate depot was determined by small angle oscillatory shear measurements presented in The in vitro dissolution results of the silica matrix and resulting triptorelin release are shown in ® are shown in Blood samples of the animal groups  were taken for bioanalysis of triptorelin and testosterone concentration in the circulation. The pharmacokinetic profiles for the administered silica-triptorelin acetate depot and Pamorelin® showed an initial burst during the first 24 h for both groups I and IV. At the 4-h time-point, the measured triptorelin concentrations for Pamorelin® were ca. 7-fold higher compared to animals that were administered the silica-triptorelin acetate. In addition, the triptorelin plasma concentrations with groups II and III remained stable for the first 2 weeks, and on average higher, compared to groups I and IV, which received Pamorelin®. After 2 weeks, the plasma concentrations of triptorelin steadily decreased for each animal group. For 42 days Group III, which received 200 µL injections of the silica-triptorelin acetate depot, showed comparable triptorelin plasma levels as Groups I and IV. Group III also showed triptorelin plasma levels above 0.2 ng/mL (LLOD) up to day 91. In contrast, the lowest triptorelin concentrations were seen for group I, that received 200 µL injections of Pamorelin®. A noteworthy observation is the pharmacokinetic similarity between groups II and IV, which received 100 µL injection of the silica-triptorelin acetate depot and the 400 µL injection of Pamorelin®, respectively. Arguably, with the silica-triptorelin acetate depot an equivalent drug exposure may be achieved with less triptorelin than Pamorelin® contains . In comparison, the triptorelin release from Pamorelinains see . One rat®, the therapeutic levels of plasma testosterone were maintained up to day 91 . The triptorelin content in the Pamorelin® remnants were 7 µg for the lower dose animal and 27 µg and 36 µg for the higher dose animals. In relation to the calculated total content in the injections the amount of triptorelin in the Pamorelin® remnants were 0.6%, 1.2% and 1.7%, respectively. The remaining average amount (n = 6) of triptorelin in the lower dose injections of silica-triptorelin acetate depot, was 85 µg, corresponding to ca. 6.7% of the calculated total content. The average value (n = 5) for the higher dose injection group of silica-triptorelin acetate depot was 263 µg of triptorelin, corresponding to ca. 10.3% of the calculated total content of triptorelin.After sacrificing the animals on day 91, remnants of silica-triptorelin acetate depots were observed and collected from 11 out of 12 animals  and Pamorelin® as more animals had remnants of the silica-triptorelin acetate depot at the end of the study. The degradation pathways of the test items are different as Pamorelin® is a PLGA-based material that typically undergoes degradation by hydrolysis and cleavage of the polymer backbone into oligomers and then to monomers. The degradation and drug release from PLGA-based formulations are a combination of drug diffusion, surface, and bulk erosion, which involves swelling of the polymer matrix as liquid  penetrates the matrix [The silica-triptorelin acetate is a longer lasting release formulation compared to Pamoreline matrix . In cont® remnants were more difficult to remove as the injected microparticles were spread-out in the tissue. Therefore, the remaining Pamorelin® was difficult to collect completely, which can also explain why less triptorelin was measured for the Pamorelin® remnants compared to the silica-triptorelin acetate depot remnants. This structural difference of these two test items can also explain, why the silica-triptorelin acetate depot remnants were observed to degrade slower compared to Pamorelin®—a single solid entity has less reactive surface compared to separated microparticles.The silica-triptorelin acetate depot remnants were observed as single solid entities, which could be removed accurately . The Pam® in the treatment of hormone-dependent prostate cancer. The developed silica-triptorelin acetate depot showed promising pharmacodynamic and pharmacokinetic results in the rat and further development of the silica-triptorelin acetate depot could yield a promising alternative for Pamorelin® that is administered using a 20 G needle. The remnant analysis showed that the hydrogel-based dosage form of silica-triptorelin acetate depot retained its 3 D structure in tissue and it was not spread-out as the microparticles of Pamorelin®.It was shown by pharmacodynamic effect (suppression of plasma testosterone levels) that triptorelin acetate retained its biological activity after manufacturing the silica-triptorelin acetate microparticles and embedding said microparticles into a silica hydrogel. The resulting silica-triptorelin acetate depot was injectable with 23 G and 25 G needles, which is beneficial in terms of injectability, and patient compliance compared to Pamorelin® injections. The silica-triptorelin acetate depot also showed long lasting triptorelin release comparable to the commercially available Pamorelin®.In vivo pharmacokinetics showed that on average injections of silica-triptorelin acetate depot formulation gave 5-fold lower Cmax values than the corresponding Pamorelin


RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites , transcript 3′ ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies. Removal of ribosomal RNAs, a major constituent (over 90%) of cellular RNA is a critical experimental step for transcriptomic studies that deal with messenger RNAs. In this manuscript, we describe a robust method to subtract ribosomal RNA from various RNA samples. The method is based on the enzymatic degradation of target RNA by short complementary DNA and RNA:DNA duplex specific nuclease. The method comprises carefully designed experimental procedures to minimize experimental bias and unwanted removal of messenger RNAs. We validate the method on various types of transcriptomic studies for seven diverse bacterial species. This method successfully removed ribosomal RNA with over 99% of efficiency and it was comparable to commercial systems even for degraded RNA samples at a fraction of a cost. PLOS Genetics paper.This is a Genetic information encoded in the genome is transferred to proteins via messenger RNAs (mRNAs). Thus, investigating mRNAs is a central approach to elucidating the fundamentals of cellular functions. Multiple techniques such as quantitative polymerase chain reaction (qPCR), microarray, and RNA sequencing (RNA-Seq) have been developed to quantitatively measure mRNAs inside a cell or their changes in response to a variety of environmental and genetic perturbations ,2. SinceSeveral methods for removing bacterial rRNAs have been developed and commercialized. Subtractive hybridization with nucleic acid probes that are reverse complementary to rRNAs is the most popular method that has been commercialized in multiple systems, such as Ribo-Zero, MICROBExpress, RiboErase, and RiboMinus –7. HowevRibonuclease H (RNase H) is an endoribonuclease that specifically digests RNA strands of RNA:DNA hybrids. The RNase H-based selective digestion of rRNAs has been proposed as a cost-effective alternative method for depleting cellular rRNAs because of its high removal efficiency ,13. An RFig 1). First, the RNA sample was denatured at 95°C for only 1 s. Prolonged incubation at a denaturing temperature that may induce spontaneous hydrolysis of RNA was not necessary for all tested samples in this study. Next, we performed RNase H reaction at an elevated temperature (65°C) to prevent unwanted hybridization of anti-rRNA oligonucleotide probes (ArOPs) to mRNA. To support this reaction, Hybridase thermostable RNase H, that has an optimum reaction temperature at 65°C or higher, was used. Synthetic single-stranded deoxyoligonucleotides with melting temperatures higher than 68°C were used as ArOPs to support efficient hybridization at a high reaction temperature (65°C).Selective digestion of rRNAs using RNase H depends on hybridization of the short deoxynucleoside probes specific to rRNAs and digestion of the RNA:DNA hetero-duplex by RNase H. Thus, minimization of the nonspecific hybridization of the probes to other RNA species is critical for efficient and nonbiased transcriptome studies. Thus, the hybridization reaction of the Ribo-Zero method is carried out at a high temperature (68°C) to reduce nonspecific hybridization. To this end, we aimed to optimize the experimental conditions in the RNase H-based rRNA depletion method to prevent unwanted RNA degradation by nonspecific binding (Methods). The probes were designed to bind approximately every 40 bp of rRNA and hybridized to rRNA in an RNA sample that had been denatured. Then, the RNA strand of RNA:DNA hetero-duplexes were subjected to RNase H digestion. Compared to the previous approaches, that use mild temperatures (22–45°C) for the digestion reaction [2+), which is required for RNase H activity [2+ concentration, which is 6.4-fold lower than that in previously reported methods [S1 Fig), which can be a useful checkpoint before proceeding to downstream study. Subsequently, ArOPs were removed by DNase I digestion followed by column-based purification, to collect remaining mRNAs. This additional enzymatic digestion can be avoided when the 3′ end of ArOP is blocked by C3-spacer or dideoxynucleotide modification, because they are not used as a substrate by ligases and polymerases; thus, they do not interfere with downstream library preparation.We used 32 nt-long single-stranded deoxyoligonucleotides as ArOPs that can bind to rRNA. When the melting temperature of the probe is lower than 68°C, the position of the probe is moved to the rRNA until the melting temperature reaches 68°C. However, when an appropriate probe could not be found or the adjusted probe overlapped with the adjacent probe, the length of the probe was shortened to 28 nt or extended up to 43 nt , and RiboErase. Without rRNA depletion, rRNA comprised over 97% of the total sequenced reads . Commercial kits showed successful removal of rRNA, except for the MICROBExpress method that showed low removal efficiency as reported elsewhere  [S1 Table) removed rRNAs from the E. coli total RNA samples at a level down to 1.63%, which is comparable to commercial methods at a fraction of the cost . Furthermore, the RiboRid method successfully depleted coverage from the entire 16S and 23S rRNA genes . Technical mock samples that underwent the same RiboRid process with nuclease-free water instead of the Hybridase enzyme, showed virtually no difference to untreated control. In addition, we inspected RNA-Seq profile of highly expressed genes to examine enzymatic reaction introduced biases or RNA degradation as reported earlier [S2 Fig). In addition, we did not observe non-specific enrichment of short rRNA reads that has been reported in the previous enzyme-based rRNA depletion approach  [S4 Fig). The RNA-Seq dataset prepared from two different biological replicates using the RiboRid method showed high reproducibility, with a correlation of 0.977 (Pearson’s R2).We first examined the capability of the RiboRid method for removing rRNAs from 500 ng total RNA samples from  ,16. The  earlier ,18, and  . Next, wThe number of genes observed by RNA-Seq was also significantly increased by rRNA removal. From the RNA-Seq result of Ribo-Zero, RiboErase, or RiboRid treated samples, expressions of 4,201, 4,166, and 4,122 genes were observed, respectively, while only 2,738 genes were detected in the control samples.S5 Fig). In contrast, samples prepared using Ribo-Zero, RiboErase, and RiboRid had identical distribution of experimental variations. Moreover, the average coefficient of variation  of the control samples was more than 3.6-fold higher than that of the rRNA-depleted sample, while the three methods had similar values. Considering the number of genes detected and the distribution of experimental variances, rRNA depletion greatly reduced the counting error of RNA-Seq, especially for genes with low abundance.Furthermore, by the nature of the Poisson process of RNA-Seq, gene expression levels measured by multiple replicates showed heteroscedasticity; that is, the variance of gene expression measured between replicates increases as the expression level decreases ,20. The Fig 2C). In addition, correlations between these technically replicated samples were higher than 0.989 . This shows that the method is sufficiently robust to accommodate practical variations in biological experiments.Next, we tested RNA input up to 1 μg and different ratios of ArOPs to total RNA. There was virtually no difference in the rRNA content of sequenced reads, indicating that the rRNA depletion method works for various amounts of input RNA and ratios of ArOPs to RNA . Although the RiboRid method was able to remove rRNA, the fraction of reads from rRNA increased to 27.4% because of the suboptimal reaction temperature of Hybridase. In addition, correlations of the samples with other technical replicates were relatively low (Pearson’s R2 = 0.744 ± 0.019) . More importantly, we predicted 46 nonspecific bindings of ArOPs to mRNA  (S2 Table). Eighteen of the probes had consecutive matches longer than 15 bp. Expression of the 18 genes measured by the RNA-Seq library prepared from the rRNA-subtracted RNA sample using the RiboRid method at 45°C was significantly underestimated when compared to the normal RiboRid-treated sample  . In contrast, we did not observe any significant difference in gene expression between untreated, mock, and RiboRid treated samples . This illustrates the importance of a high reaction temperature in preventing nonspecific binding of probes to mRNA and demonstrates that high reaction temperature of RiboRid was able to avoid experimental bias.To examine the possible nonspecific binding of ArOPs to mRNA, we repeated RNA-Seq from the same RNA sample using the RiboRid method at 45°C . Although gaps in K. oxytoca rRNAs, where no ArOP hybridizes, span a maximum length of 163 nt, 76 ArOPs were dense enough to remove rRNAs to a level less than 3% of mappable sequence reads in K. oxytoca RNA-Seq, with a correlation of 0.998 (Pearson’s R2) between biological replicates (Table 1). rRNA removal of K. oxytoca using anti-E. coli ArOPs indicates that the number of ArOPs may be reduced to a much lower density. Thus, we designed a new probe set in which each probe was spaced at a longer distance. The new probe set consisted of 55 probes that could hybridize approximately every 80 nt on the rRNAs (S3 Table). We also adjusted the protocol further to reduce the cost and time of the RiboRid method. First, ArOPs were synthesized with a C3 spacer at the 3′ end, which is not utilized as a substrate for ligases and polymerase in downstream library construction. Originally, oligonucleotide probes remained after the RiboRid reaction needed to be removed by DNase digestion, since they can interfere with the downstream PCR amplification step of sequencing library construction. However, C3 spacer-modified oligonucleotides cannot be used as primers for DNA polymerase; thus, DNase I digestion of probes at the end of the RiboRid method can be avoided. Alternatively, the 3′ end of pre-existing probes can be blocked by attaching dideoxynucleotide analogs using terminal deoxynucleotidyl transferases instead of C3 spacer modification. Then, the column-based clean-up step was replaced by SPRI paramagnetic bead purification. Together with these modifications, the cost and processing time of the RiboRid method can be reduced considerably (S1 and S2 Protocol) without increasing the rRNA fraction or experimental bias .Next, we applied the method and the same ArOPs to RNA-Seq of Pseudomonas aeruginosa and Bacteroides thetaiotaomicron, and two Gram-positive bacteria, Eubacterium limosum and Staphylococcus aureus, with ArOPs specific for each bacterium (Table 2). P. aeruginosa is a Gram-negative opportunistic pathogen that is a common cause of pneumonia and other infections in various parts of the body [Fig 4A). There were large amounts of RNA fragments in the intergenic region of the rRNA operons. Considering rRNA maturation and processing in bacteria, the fragments were predicted to be the pre-rRNAs that were not depleted due to the absence of complementary ArOPs. The pre-rRNAs comprised an average of 0.84% of the mapped reads in RNA-Seq. Although the amount of pre-rRNA reads was negligible when compared to mRNA reads, we tested two different approaches to further remove pre-rRNA species. First, a two-fold increase in Hybridase in the reaction did not affect the overall amount of non-mRNA or pre-rRNA reads . In fact, supplementation of a few anti-pre-rRNA oligos (S4 Table) in the Hybridase reaction reduced the amount of pre-rRNA fragments from the RNA sample to 0.06% .To examine the versatility of the RiboRid method for diverse bacterial species, we applied RiboRid to two Gram-negative bacteria, the body . rRNAs wthe body  using thS. aureus, a common pathogen that is one of the biggest threats to the health care system due to the emergence of strains with antibiotic resistance [E. limosum, a model strain of CO2-fixing acetogenic bacteria [Table 2). The fraction of S. aureus rRNAs in RNA-Seq was lower than 1.72% regardless of the culture medium, with Pearson’s correlation (R2) between biological replicates higher than 0.955 . In particular, rRNAs comprised only 0.02% of the total mapped reads when RNA was prepared from the cells grown in RPMI medium. The RiboRid method was also capable of reducing rRNAs from E. limosum down to 2.25% with high reproducibility (R2 = 0.949) between biological replicates (Table 2).We further applied the RiboRid method to two Gram-positive bacteria: sistance ,24; and bacteria . rRNAs fB. thetaiotaomicron is one of the major constituents of the human gut commensal microbiome [B. thetaiotaomicron. In detail, rRNA from the RiboRid-treated sample comprised 27.20 ± 5.03% (Table 2), although the RNA expression measured from RiboRid-treated sample was highly correlated with that of Ribo-Zero-treated samples with a Pearson’s correlation (R2) of 0.915 ± 0.01 . To improve the efficiency of rRNA removal, we designed a new ArOP set (ArOP Set 2) with different DNA sequences that have higher melting temperatures, so that they can hybridize to rRNA stronger at the elevated reaction temperature of RiboRid. Although the fraction of rRNA to the total mapped reads was reduced to 17.82 ± 0.05%, its correlation with the Ribo-Zero- or standard ArOP (ArOP Set 1)-treated sample was decreased to 0.741 (R2) . Lowering the reaction temperature to 58°C, which is at least 10°C lower than the annealing temperature, did not improve the rRNA removal efficiency (Table 2). A recent report also indicated that both Ribo-Zero and RNase H-based removal of rRNAs Bacteroidetes dorei, were relatively inefficient, possibly due to its high genomic AT-content [Bacteroidetes; however, the RiboRid method was able to remove rRNAs to a level less than 20% of total RNA.crobiome ,27. Unli-content . As illu-content , furtherS. coelicolor, which is a Gram-positive soil bacterium well-known for producing various secondary metabolites [Table 3). To address possible non-specific random RNA degradation during the RiboRid treatment that may decrease resolution of single nucleotide-sensitive study such as Term-Seq, we inspected profile of Term-Seq . For example, transcript 3′ end detected from two biological replicates coincides with each other in nucleotide-level . Furthermore, the Term-Seq signal from two replicates were highly correlated (Pearson’s R2 of 0.9998) across the entire genome . Thus, we concluded that RiboRid did not introduce any noise for the transcriptomic study. With successful depletion of rRNAs from total RNA of S. coelicolor with high GC content (72.4%), we were able to identify a conserved rho-independent transcriptional terminator motif at the 110 transcript 3′ ends, which resembles the rho-independent transcriptional terminators of the closely related actinomycetes, S. lividans  [Fig 5B) [To further validate the RiboRid method for other challenging transcriptomic analysis methods, we conducted Term-Seq . The Terabolites . Reads m . In addi[Fig 5B) . This deE. coli using the RiboRid method, which was modified by adopting a previously developed streamlined protocol (see Methods) [Table 3). Close inspection of the sequencing profile indicated that specific regions of rRNA were enriched . Thus, we designed and supplemented six additional probes  targeting the enriched regions (S5 Table). The specific RNA fragments were clearly depleted after RiboRid treatment with the additional probe set 1 . However, another rRNA fragment remained and became more noticeable relative to the fragments targeted by probe set 1. We constructed six additional probes  and RiboRid with the two additional probe sets reduced the fraction of reads mapped on rRNA down to the level shown by the Ribo-Zero-treated sample, which was approximately 70% . Meta-analysis of the ribosome profile on coding sequences showed a nucleotide-resolution profile of ribosome footprint and pausing on the start codon , which is comparable to Ribo-Zero treated profile , illustrating the capability of RiboRid in ribosome profiling [Ribosome profiling is a transcriptome sequencing technique that surveys mRNAs that are actively translated by capturing ribosome-protected fragments (RPFs) . It is oMethods) . Initialrofiling .Taken together, RiboRid effectively removed the fraction of rRNA even from highly fragmented polysomal RNA samples, indicating that it can be used for complicated transcriptomic studies other than RNA-Seq. The single base-pair resolution required for probing 3′ end information and the precise polysomal location on the RNA were not compromised by the method developed in this study.Based on the transcriptomic sequencing results presented in this study, we demonstrated a strategy for effectively depleting rRNA from bacterial RNA samples. The RiboRid method is highly advanced and effective method, capable of removing rRNA from highly fragmented RNA samples without losing precise biological information of the mRNA. Owing to thorough experimental and protocol design considerations regarding hybridization temperature and probe design, the prevalent rRNA species in bacterial RNA samples could be efficiently removed without introducing any experimental bias or causing nonspecific mRNA removal. Furthermore, a short incubation time at denaturing temperature, low concentration of divalent magnesium cations, and mild pH prevented RNA degradation observed in the previous enzymatic approaches ,18. MoreS. aureus sample. However, in the case of B. thetaiotaomicron, rRNA comprised 17% of all mappable rRNA reads. This is possibly because of the high AT content of the organism, in which hybridization of the probe at high temperature may be inefficient. However, this can be easily compensated for by using a slightly higher sequencing depth, as the sequencing cost is much lower than the difference between the costs of the RiboRid and commercial methods.RiboRid is highly efficient and could remove rRNA with an efficiency of up to 99.99% in the https://github.com/SBRG/RiboRid_Design); this script can design a probe set for any custom genome or rRNA sequence. In addition, we designed and deposited probe sets for representative bacterial species available in the RefSeq database . This method provides a cost-effective, rapid, and powerful alternative means to deplete rRNA that outperforms previously developed and reported methods. In particular, this method is valuable for routine large-scale transcriptome studies and reduces the burden of high-cost commercial kits.To facilitate the use of this method, a step-by-step protocol is available in the Escherichia coli MG1655, Klebsiella oxytoca KCTC 1686, Pseudomonas aeruginosa PAO1, and Bacteroides thetaiotaomicron VPI-5482) and three Gram-positive strains  were used in this study. E. coli was grown in Luria-Bertani (LB) broth at 37°C. K. oxytoca KCTC 1686 was grown at 37°C in T1 semi-defined xylose medium 2SO4, 8.7 g/L K2HPO4, 6.8 g/L KH2PO4, 0.25 g/L MgSO4⋅7H2O, 0.05 g/L FeSO4⋅7H2O, 0.001 g/L ZnSO4⋅7H2O, 0.001 g/L MnSO4⋅H2O, 0.001 g/L CaCl2⋅2H2O, and 20 g/L xylose). P. aeruginosa was grown at 37°C in M9 medium  supplemented with 2 g/L succinate. S. aureus was cultured in cation-adjusted Mueller Hinton Broth (CA-MHB) or Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% LB medium (R10LB). The CA-MHB contained 2 g/L beef infusion solids, 1.5 g/L starch, 17.5 g/L casein hydrolysate, 25 mg/L calcium, and 12.5 mg/L magnesium. E. limosum was cultivated at 37°C in DSM135 medium comprising 1 g/L NH4Cl, 2 g/L yeast extract, 10 g/L NaHCO3, 0.1 g/L MgSO4⋅7H2O, 0.3 g/L cysteine-hydrochloride, 10 mL vitamin solution , 4.6 mM KH2PO4, 5.4 mM K2HPO4, 4 μM resazurin, and 20 mL trace element solution 2⋅12H2O, 10 mg/L H3BO3, 10 mg/L Na2MO4⋅2H2O, 30 mg/L NiCl2⋅6H2O, 0.3 mg/L Na2SeO3⋅5 H2O, 0.4 mg/L Na2WO4⋅2H2O), supplemented with 5 g/L glucose. E. limosum was cultured anaerobically in 150-mL serum bottle containing 100 mL culture medium purged with N2 gas at a pressure of 200 kPa. B. thetaiotaomicron was cultured in brain-heart infusion-supplemented broth (BHIS). One liter of BHIS broth contains 37 g Brain-Heart Infusion , 5 g yeast extract, 0.5 g/L cysteine hydrochloride monohydrate, 0.2 mM L-histidine, 1.9 μM hemin, and 1 μg/mL menadione and was adjusted to pH 8. Cells were cultured in a 150-mL serum bottle containing 100 mL culture media purged with N2/CO2 (90:10) gas at a pressure of 80 kPa at 37°C with agitation. S. coelicolor was cultured in 50 mL R5 medium at 30°C with 8 g glass beads (diameter of 3 mm) contained in a 250-mL baffled Erlenmeyer flask. One liter of the R5 medium is composed of 5.73 g N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid , 103 g sucrose, 10 g glucose, 5 g yeast extract, 10.12 g MgCl2∙6H2O, 0.25 g K2SO4, 0.1 g casamino acids, 0.08 mg ZnCl2, 0.4 mg FeCl3∙6H2O, 0.02 mg CuCl2∙2H2O, 0.02 mg MnCl2∙4H2O, 0.02 mg Na2B4O7∙10H2O, and 0.02 mg (NH4)6Mo7O24∙4H2O.Four Gram-negative strains  was resuspended in 100 μL RNASnap solution  and incubated at 95°C for 7 min. The resuspension was centrifuged at 16,000 × g for 5 min and RNA with a size larger than 200 nt in the clear supernatant was purified using RNA Clean & Concentrator-5 Kit  according to the manufacturer’s instructions. Total RNA of E. limosum was extracted from 100 mL mid-exponential growth culture (OD600nm = 1.5). The collected cells were resuspended in 500 μL lysis buffer  and ground using a mortar and pestle after flash freezing with liquid nitrogen. RNA was isolated from the supernatant of the ground sample using TRIzol Reagent  followed by an RNA Clean & Concentrator-5 Kit according to the manufacturer’s instructions. Total RNA of P. aeruginosa and S. aureus was extracted using Quick-RNA Fungal/Bacterial Microprep Kit (Zymo) from 3 mL culture sampled at an OD600nm of 0.4. Briefly, the cell pellet was resuspended in 800 μL RNA lysis buffer. The resuspension was then transferred into a ZR BashingBead Lysis Tube (0.1 and 0.5 mm) and homogenized in a bead beater. RNA in the cleared lysate (400 μL) was purified by column purification. Total RNA of S. coelicolor was extracted using the hot phenol method from 50 mL culture samples at the early exponential, transition, late exponential, and stationary growth phases . First, the collected cells were resuspended in Solution 1  and incubated for 10 min at 30°C. The supernatant of the mixture was removed by centrifugation, and the cell pellet was resuspended in Solution 2 . The suspension was mixed with an equal volume of phenol:chloroform (5:1) solution and incubated for 5 min at 65°C. RNA was isolated by isopropanol precipitation from the aqueous phase and separated by centrifugation.Total RNA of p method  with slihttps://github.com/SBRG/RiboRid_Design). The program can be either imported into other pipelines or executed as a standalone program from the command line. As a basic input, the script takes in GenBank files (with annotated rRNA) or a FASTA file containing the rRNA sequences for the target organism. Given the rRNA sequences in either format, the script starts by building a consensus sequences for rRNAs. These consensus sequences were then used to build a DNA probe library. To build the library for each rRNA type, we started from the 50th sequence position of the rDNA. The first probe was defined as the DNA sequence that started from this position and was the length of the user-defined probe. With a default probe length of 32, the probe starts at position 50 and ends at position 81. If the probe has a melting temperature above the defined melting threshold (68°C used in this study), then the script looks for the next probe downstream with the user-defined maximum gap (default of 50 nt) between the probes. Typically, 12–25 probes per kb of rRNA are required for efficient removal of rRNA (one probe per 40–80 nt). The melting temperatures of the probes were calculated using the OligoAnalyzer Tool . If the melting temperature of the probe is below the threshold, then the script will look for another probe up to the user-defined maximum search space (default of 10 bp) upstream of the starting search position and record the probe with the highest melting temperature. Note that the probe with the highest melting temperature was recorded even if the melting temperature of the probe was below the threshold. Following these steps, the script generates probes in a stepwise manner until all the rRNAs are covered. Once the design process is finished, the designed probe sequences are recorded in a FASTA file, and the metadata associated with each probe  is recorded in a.csv text file. Using the script, the melting temperatures of 32 nt-long probes were higher than 68°C in 98.3% of the design attempts. In a small number of cases (1.7%), the melting temperature criteria were met by manually adjusting the lengths of probes from 28 to 43 nt .The probes used were 32 nt-long deoxynucleotides reverse complementary to the rRNA with melting temperatures at least 3°C higher than the rRNA digestion reaction temperature (68°C). The anti-rRNA oligonucleotide probes were designed using an in-house Python program  prewarmed at room temperature was added. The reaction was carried out at 65°C for 20 min and 90°C for 1 s to rehybridize the ArOP to the remaining rRNA, and then at 65°C for an additional 10 min. Then, RNA larger than 200 nt was extracted using RNA Clean & Concentrator Kit. A nucleic acid-binding column was saved for later use. The remaining ArOPs were removed by DNase I treatment in a reaction composed of 10 U RNase-free DNase I and 5 μL 10× DNase I buffer in a total reaction volume of 50 μL. The DNase I reaction was carried out by consecutive 5 min incubations at 25, 30, 35, 40, and 45°C. The reaction was again purified with the RNA Clean & Concentrator Kit using a nucleic acid binding column saved from the previous clean-up. A step-by-step protocol is provided in S1 and S2 Protocol. The column-based RNA purification step can be replaced by a solid-phase reversible immobilization (SPRI) bead-based purification method. In this study, RNA was purified using 1.8 volume of CleanNGS DNA & RNA SPRI Bead  according to the manufacturer’s instructions. When using a 3′-C3 carbon spacer-modified ArOP that does not participate in downstream reactions , the DNase I treatment step for removing residual ArOPs after the hybridase reaction was not performed.Before performing rRNA depletion, RNA content was measured using a Qubit RNA HS Assay Kit (Thermo) with a Qubit 2.0 fluorometer (Thermo). RNA extract was subjected to DNase I treatment for removal of DNA contamination at 37°C for 10 min in 15 μL reaction mixture containing 0.5–1 μg total RNA, 1.5 μL 10× DNase I Buffer, and 2 U RNase-free DNase I. Then, DNase I was inactivated at 75°C for 10 min after addition of 15 μL Hybridase Complement Buffer comprising 90 mM Tris-HCl (pH 7.5) and 200 mM KCl. The ArOP mixture (1 μL) containing 5 pmol of each probe and 100 mM MgClS6 Table.RNA-Seq libraries were constructed from approximately 100 ng rRNA-depleted RNA using a TruSeq Stranded mRNA LT Sample Prep Kit  or KAPA RNA HyperPrep Kit  according to the manufacturer’s instructions. Constructed sequencing libraries were quantified using a Qubit dsDNA HS Assay Kit with a Qubit 2.0 fluorometer and a TapeStation 2200  equipped with a High Sensitivity D1000 Screen Tape (Agilent). The library was sequenced using an Illumina platform. Information on NGS (run recipe and instrument) is summarized in S6 Table.Total RNA extract containing 5 μg total RNA was treated with DNase I at 37°C for 15 min in a 50 μL reaction comprising 2 U RNase-free DNase I and 5 μL 10× DNase I buffer. The Term-Seq library was constructed as previously described . BrieflyE. coli culture was collected after 5 min of treatment with chloramphenicol (34 mg/mL) at an exponential growth phase. Cells were flash frozen with 0.5 mL lysis buffer  and lysed using a mortar and pestle. Then, the supernatant containing 100 μg RNA was treated with 2,000 gel units of micrococcal nuclease . Polysomes were recovered from MNase-digested samples using Illustra MicroSpin S-400 HR columns  followed by phenol:chloroform:isoamyl alcohol extraction. rRNA was removed from 1 μg polysome-protected RNA using a Ribo-Zero rRNA Removal Kit according to the manufacturer’s instructions. The rRNA-subtracted RNA samples were phosphorylated by treating 10 U T4 polynucleotide kinase (NEB) at 37°C for 1 h and purified with RNeasy MinElute columns . Sequencing libraries were prepared from phosphorylated RNA samples using the NEBNext Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer’s instructions. The library was sequenced using an Illumina platform. Information on NGS (run recipe and instrument) is summarized in S6 Table.Ribosome profiling was conducted using a method described in a previous report . BrieflyS6 Table.Polysomal RNA was prepared as described previously. The polysomal RNA samples were subjected to phosphorylation without rRNA removal by treating with 10 U T4 polynucleotide kinase at 37°C for 1 h and purified with RNeasy MinElute columns. 5′ sequencing adaptors were attached to phosphorylated RNA samples using the NEBNext Small RNA Library Prep Set for Illumina according to the manufacturer’s instructions. After adaptor ligation, RNA samples were subjected to a column-based RiboRid reaction with dideoxy-modified ArOP and without DNase I treatment. RNA purification steps were performed using the RNA Clean & Concentrator Kit for RNA size > 17 nt. The dideoxy-modified ArOPs were prepared by incubating 5.4 nmol (50 pmol each probes) of ArOPs at 37°C for 4 h with 50 U terminal deoxynucleotide transferase , 20 μL 5× Reaction Buffer, 20 mM ddNTP  in 100 μL reaction mixture. The modified ArOP was purified using the Oligo Clean & Concentrator Kit (Zymo) as described by the manufacturer. Then, 3′ RNA adaptor ligation, reverse transcription, and sequencing library amplification were performed using the NEBNext Small RNA Library Prep Set for Illumina. The library was sequenced using an Illumina platform. Information on NGS (run recipe and instrument) is summarized in E. coli), NC_016612 (K. oxytoca), NC_002516.2 (P. aeruginosa), NC_010079 (S. aureus DNA), NC_010063 (S. aureus plasmid pUSA300HOUMR), NC_012417 (S. aureus plasmid pUSA01-HOU), NC_004663 , NC_004703.1 (B. thetaiotaomicron plasmid DNA), NZ_CP019962.1 (E. limosum), and NC_003888.3 (S. coelicolor). Gene expression levels were calculated by counting strand-specific reads of genes (antisense) and normalized using DESeq2 [−55 and each sequence aligned to the motif with a p-value lower than 1.32 × 10−4. The meta-analysis of ribosome profiles was performed using the STATR pipeline, as described previously [Sequencing data were processed on a CLC Genomics Workbench . Raw reads were trimmed using the Trim Sequence Tool in NGS Core Tools with a quality limit of 0.05. Reads with more than two ambiguous nucleotides were discarded and the quality trimmed reads were mapped on either the reference genome or rRNAs using the following parameters: mismatch cost of 2, indel cost of 3, and similarity and length fraction of 0.9. Reference genome sequences were downloaded from NCBI under the accession numbers NC_000913.3 (g DESeq2 . Nonspecg DESeq2 . The foueviously . Wilcoxoeviously S1 Protocol(PDF)Click here for additional data file.S2 Protocol(PDF)Click here for additional data file.S1 Fig(A) Total RNA sample used. Bands of 16S and 23S rRNA are annotated. (B) RNA sample after RiboRid treatment. There was no observable rRNA and significant degradation of mRNA.(PNG)Click here for additional data file.S2 FigrpsA gene and (B) pdhR-aceEF-lpd operon. (C) Pairwise comparison of cumulative read count of RNA-Seq profiles on rpsA gene.RNA-Seq profiles on (A) (PNG)Click here for additional data file.S3 FigExpress. Read length distributions of (C) rRNA reads and (D) non-rRNA reads detected by RNA-Seq of RNA samples prepared by Ribo-Zero, RiboErase, or RiboRid.Read length distributions of (A) rRNA reads and (B) non-rRNA reads detected by RNA-Seq of RNA samples without rRNA removal or treated with MICROB(PNG)Click here for additional data file.S4 Fig2) between samples and biological replicates. (B) Scatter plots showing mRNA expression level measured by RNA-Seq from different rRNA depletion methods. Each circles indicate individual genes.(A) Pairwise Pearson’s correlations (R(PNG)Click here for additional data file.S5 Fig2(expression level+1)). Rank indicates rank of mean expression in an ascending order . The orange lines show moving averages of standard deviation with a window size of 50 genes. n: number of genes detected. Average CV: average coefficient of variation (standard deviation divided by mean expression of a gene) of detected genes.Mean and standard deviation are calculated from log2 transformed pseudo-count (Log(PNG)Click here for additional data file.S6 Fig(A) Reproducibility of RNA-Seq with different combination of input RNA, amount of ArOP used, and reaction temperature of performing RiboRid. (B) Pairwise comparison between gene expression levels measured by two different technical replicates of RNA-Seq results. (C) Reproducibility of RNA-Seq prepared from the standard RiboRid method using column or the method with C3 spacer-modified ArOPs and SPRI bead-based purification.(PNG)Click here for additional data file.S7 FigE-value; BLASTN) were compared as a group. E<1; ≥15 nt: genes with E-value lower than 1  and with 15 nt or more consecutive matches. ns: difference between fold-change distributions are not significant (Wilcoxon’s rank-sum test). Box limits, whiskers, and center lines indicate 1st and 3rd quartiles, 10th and 90th percentiles, and the median of the distribution, respectively. Dots are individual genes. Number of subjected genes are indicated above the graph. ns: not significant (Wilcoxon’s rank-sum test).Genes with different degree of sequence similarity ((PNG)Click here for additional data file.S8 Fig2) between biological replicates of (A) P. aeruginosa, (B) S. aureus, and (C) B. thetaiotaomicron.Pairwise Pearson’s correlation (R(PNG)Click here for additional data file.S9 Fig2 of 0.9998) throughout the genome.Term-Seq profiles on genes encoding ribosomal protein (A) RplT and (B) RpmJ. The two biological replicates correlate to each other with a single nucleotide precision without any noticeable noise. (C) Pairwise comparison of Term-Seq signal from the two biological replicates showed linear correlation (Pearson’s R(PNG)Click here for additional data file.S10 FigEither 5′ or 3′ ends of sequencing reads were used to determine boundary of ribosome. Ribosome density (RD) is average ribosome profile of coding sequences normalized by dividing ribosome profile of each positions with the maximum peak height in 400 nt window.(PNG)Click here for additional data file.S1 TableSE: single-ended recipe. PE: pair-ended recipe.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table3SpC3: C3-spacer modification at the 3′ end.(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


As one remarkable finding, the potential cross-kingdom transfer of small RNA to animals via food is introduced, although this Research Topic remains controversial and inconclusive. The function of these RNA species is described as epigenetic inheritance, cleavage of target mRNA–RNA interference, and translational repression. Among them, it is worthwhile to emphasize the use of small RNA-delivered via EV for the control of pathogen infection. Yu et al. addresses another historically controversial Research Topic such as the systemic signaling that controls flowering. Different studies have revealed that the Flowering locus T (FT) protein is a major component of FT flowering promotion. However, several groups including the authors' group have observed mobility of FT mRNA or FT mRNA localization near the plasmodesmata in expressed cells as a related phenomenon of symplasmic transport. This mini review summarizes a series of experiments, such as virus-based system, grafting experiments, and intracellular RNA imaging, and discusses potential non-cell autonomous behavior of FT mRNA. In addition, as a potential biotechnological tool, the addition of FT mRNA sequence to guide RNA is proposed to overcome virus exclusion from the shoot apical meristem to generate heritable virus-induced gene/genome editing. Since gene editing will be a key technology to improve and accelerate agricultural breeding, such an RNA delivery technique based on an evolutionarily conserved RNA transport system is an attractive catalyst for new opportunities to face the challenges posed by global climate change to sustainable and robust agriculture. Lezzhov et al. contributed an opinion article about phloem exit as a possible control point in selective systemic transport of RNA. According to these authors, this selective transport would rely on the presence of “phloem transport signals” (PTS) that would be bound by PTS-binding proteins involved in selection of RNA molecules for exit to surrounding tissues. Conceivably, the existence of non-identical PTS-binding proteins in different plant organs would explain the fact that distinct subsets of mobile mRNAs are delivered to different target organs. The authors also discuss potential involvement of the endoplasmic reticulum and actin network in RNA transport in sink-to-source manner, opposite the direction of phloem flow. Vargas-Hernández et al. and Calderón-Pérez et al. provide original studies related to this field. Vargas-Hernández et al. provide a study on ANAC087, a homolog of CmNACP1, the mRNA of which has been shown to move long-distance through the phloem in Cucurbita maxima (pumpkin) and through a graft junction. By taking advantage of the model plant system, Arabidopsis, the authors address the function of CmNACP1 homologs. Out of six Arabidopsis homologs, the authors focused on ANAC087 because it possesses the highest sequence similarity. The promoter GUS analysis of ANAC087 showed that strong GUS expression was found in leaf vascular tissue and stem as well as the shoot apex, roots and the base of the trichomes. Although the mobility of ANAC087 mRNA has not been examined by the authors, the biological relevance to leaf senescence and aerial rosette formation was demonstrated by overexpression of ANAC087. Calderón-Pérez et al. demonstrated the potential use of the plant macromolecular trafficking system to mobilize antimicrobial peptides (AMPs). The authors utilized the knowledge that a pumpkin phloem mobile protein, CmPP16, facilitates non-specific RNA transport. It has been thought that CmPP16 regulates plasmodesmata permeability and facilitates the systemic movement of other molecules. The authors identified the citrus homolog, named CsPP16, and engineered transgenic citrus trees harboring CsPP16-fused AMPs. Translocation of the CsPP16-fused AMPs into sieve elements was confirmed in these transgenic citrus plants. Finally, the authors demonstrated the positive effect of the engineering on reduction of live bacterial infection for a period of 1 year. Moreover, further significant reduction was observed when AMPs were combined. This study demonstrated the possible combinational usage of antimicrobials and macromolecular trafficking system to control bacterial infection.via phloem, e.g., RNA-protein complex, the manner of selective transport at the loading and unloading sites, and the cellular translocation pathways between different tissue types, will provide knowledge on their roles in plant biology and also enhance bioengineering techniques for crop improvement.The research articles featured in this Research Topic cover a range of small RNA, mRNA, viral RNA, and components of RNA transport. This Research Topic further advances our understanding of RNA systemic transport and its potential use of bioengineering. Emerging findings and hypothesis need further experimental proofs in future studies. Further studies of RNA trafficking mechanism, such as the molecular forms of trafficking MN drafted the manuscript. VP, JQ, and XW edited and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


N = 305) reported how many times they lied in the past 24 hours and answered personality questions. Results indicate that the few prolific liars pattern is evident in Japan thereby advancing TDT. Results also show that Japanese frequent liars tend to have Dark Triad personality traits, but the nature of the findings may be unique to Japan. Results of the generalized linear model suggest that the Dark Triad components of Machiavellianism and psychopathy exacerbate lying behavior by reducing the guilt associated with lying. However, narcissism encourages guilt and therefore inhibits lying behavior with both direct and indirect effects. These narcissism findings appear to contradict prior studies but stem from use of a more appropriate statistical analysis or the Japanese context.Truth-Default Theory (TDT) predicts that across countries and cultures, a few people tell most of the lies, while a majority of people lie less frequently than average. This prediction, referred to as “a few prolific liars,” is tested in Japan. The study further investigated the extent to which the Dark Triad personality traits predict the frequency of lying. University students ( Most people lie at least once in a while, but how often people lie is frequently misunderstood. For example, DePaulo et al.  used a dTruth-Default Theory , 4, howeTDT is a pan-cultural theory. TDT’s core presumptions concern human nature and are not tied to any particular culture, religion, government, or social structure. According to TDT, the prevalence and distribution of lying in communication are critical considerations because prevalence rates have implications for the utility of the truth-default. Truth-default is the idea that people passively and uncritically accept the content of incoming messages as honest and truthful unless some trigger event prompts suspicion –5. The tFinding infrequent lying is important to TDT because humans tend to believe others. Most often people are in a truth-default state in which they passively believe others and where thoughts about deception do not even come to mind. Even when suspicion is triggered, people still tend to be truth-biased. Believing others through the truth-default and truth-bias is advantageous if more people are honest  , 6.Initial support for the few prolific liar prediction from TDT was reported by Serota et al.  who survThe phenomenon of a few prolific liars appears to be broadly recurring and TDT’s predictions are pan-cultural . HoweverExtending beyond TDT predictions, we additionally investigated the personalities of prolific liars. TDT’s specification of a few prolific liars implies that there are individual differences in the proclivity to lie. If this is the case, then this individual variation may be explainable in terms of various personality traits and dimensions. Although numerous traits are associated with individual differences in communication, the Dark Triad psychological traits seem especially relevant due to their “dark” nature. The Dark Triad refers to socially aversive personality traits that include Machiavellianism, psychopathy, and narcissism . Prior sBesides Dark Triad, we wondered if other personality traits investigated by previous studies might mediate the effects of personality (the Dark Triad) on lie frequency. For example, Halevy et al.  indicateThis study employs a generalized linear model (GLM), which is useful when a dependent variable is not normally distributed. In this study, we investigated the personality traits of a few prolific liars—including the Dark Triad—by using GLM while controlling other variables to replicate the long-tail phenomenon of a few prolific liars.This study was approved by the Osaka University’s School of Human Sciences’ ethics committee (HB300-07). All participants provided written consent on the survey page.Mage = 19.61 ±3.33 years) at two Japanese universities. The first author called on university students to voluntarily answer the survey after a colleague’s class. Only students who agreed to answer participated in our study. Participants were excluded if they reported that they did not understand the instructions of this survey (5 participants), or marked a wrong number on the DQS , or their first language was not Japanese (23 participants), or if they reported over 10,000 lies in the past 24 hours (1 participant). Several participants violated more than one of these criteria. After these exclusions, a total of 305 participants  were included in the analyses. This sample size provided statistical power of .94 to detect a zero-order correlation of r = .20 at p < .05.The initial sample included 340 undergraduate students  . The DTDTo exclude random and biased responses, the personality traits questionnaire also included the directed questions scale (DQS)  and the At the end of the survey, participants provided demographic information and indicated confidence in the accuracy of their lie responses. Participants used a five-point scale to answer, “How accurate do you think the number of lies you reported was?” This question was added to confirm that the few prolific liars phenomenon is not merely an artifact of guessing as some participants might not remember how many times they had lied in the past 24 hours. The aim of this check was to investigate whether the phenomenon replicates even when we exclude unsure participants’ answers.SD = 9.50, Median = 1.00, Range: 0 to 150, Mode = 0) during the prior 24-hour period was reported. The mode was zero (37.4% of participants reported that they did not lie in the past 24 hours), and 51.5% reported that they told one to five lies. Only 11.1% reported six or more lies; but this group accounted for 59.3% of total reported lies (536 out of 904 lies).The number of lies in the past 24 hours were calculated and graphed see . Prior stotal study participants (including those who reported one or more lies in the past 24 hours) reported lying in the preceding month, consistent with monthly rates observed in United States studies  = .14, p = .012) and marginally correlated with Machiavellianism . For narcissism, however, there was no significant correlation with lying frequency . In addition, guilt  was negatively correlated while perceived detection ability  had a marginal correlation with lying frequency.To examine the personality traits of prolific liars, we calculated the zero-order Pearson’s correlations among the variables measured  and narcissism  as well as psychopathy  was positively correlated with lying frequency. In addition, perceived detection ability did not have a significant correlation with lying frequency , while guilt  and acceptance  had significant correlations.Since the distribution of lying frequency was very skewed, we also calculated Spearman’s rank correlations  when controlling Machiavellianism and psychopathy. While the zero-order correlations of narcissism include the effects of Machiavellianism and psychopathy, the result of the negative binomial regression partials out the effects of them when assessing the effect of narcissism. Thus, it may be safe to say that the negative coefficient of narcissism is the pure effect of narcissism on lying frequency. This may be the reason why we had the negative coefficient while we had a positive correlation between lying frequency and narcissism in Spearman’s rank correlation.This negative effect of narcissism on lying is interpretable from three perspectives. The first is narcissism’s relative brightness. Narcissism is considered the least dark trait among the three . NarcissThe second consideration is lying types. Our study did not classify lying types, so all kinds of lies are included in the analysis. Narcissists are thought to tell lies mostly about themselves to make a good impression on others. In fact, Jonason et al.  revealedThe third possibility is cultural differences. Narcissism scores may differ across countries. Foster et al.  found thFurther research on the subtypes of narcissism also might be useful for interpreting this result. Narcissism can be divided into vulnerable narcissism—associated with introversion, defensiveness, anxiety and vulnerability to life’s traumas—and grandiose narcissism—associated with extraversion, self-assurance, exhibitionism, and aggression . PreviouThe current study has three limitations to consider. First, our analysis did not control for the frequency of social interaction. The Dark Triad personality traits are positively correlated with extraversion among the Big Five personality traits . Thus, aSecond, the results of this study are based solely on lies reported by college students. To improve the generalizability of the results, a study obtaining lie reports from a broader sample could be conducted. Fortunately, research in other countries is informative about how student samples are similar and different from more broadly representative samples. Research has documented the few prolific liars pattern  in studies of both students and adult samples , 8, 10. Third, the measurement of the Dark Triad used in this study may be insufficient. The Japanese version of the DTDD has differences from the original English version . The differences are most evident in Machiavellianism and psychopathy, but due to the strict translation procedures they are not substantial. It appears unlikely that the divergence for narcissism may have resulted from a translation problem.Future research might examine other TDT propositions in Japan and other countries in Asia. Truth-bias has been documented in Korea  and MuraOverall, this research clearly indicates the existence of a few prolific liars in a student sample in Japan. As observed in other parts of the world, most Japanese people tell few or no lies on a given day and a small number of people, prolific liars, tell the majority of lies. Additionally, the study found that lying frequency increased with higher Machiavellianism and psychopathy scores, and that these factors are mediated by feelings of guilt. Documenting the mediating effects of guilt expands our knowledge about lying and its prediction. This mediating effect suggests that people with certain personality traits such as Machiavellianism may feel less guilty about lying and consequently have fewer inhibitions about lying. Practically, it may be effective to activate people’s feelings of guilt to suppress lying in real world. We further observed an unexpected effect of narcissism, which inhibited lying frequency. How narcissism affects lying should be investigated further.S1 File(CSV)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Non-Coding RNA Scientific Board wishes you an exciting and fruitful read.We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently. We hope to keep you up-to-date with non-coding RNA research works that are outside your study area. The In a finding that has elated and perplexed researchers, a recent study has shown circulating determinants as predictive biomarkers for end stage kidney disease (ESKD). A key objective—because not all diabetic patients progress identically—is the early prediction of diabetic kidney disease (DKD). To address this unmet need, the study has assessed the expression of circulating miRNAs and proteins in peripheral blood from individuals with DKD (type 1 and type 2 diabetes) from the Joslin Kidney Study (88% of European–American ancestry) and the Pima Indian Kidney Study (100% of Indigenous–American ancestry) [MALAT1 is one of the most abundant and highly conserved lncRNAs and contributes to various features of malignancy, as proliferation and migration. Subnuclear localization and activity of MALAT1 are controlled by various protein factors. The N6-methyladenosine (m6A) writer METTL3 for example methylates MALAT1 lncRNA leading to its stabilization, while the m6A reader YTHDC1 binds to m6A-modified MALAT1 and regulates the composition/function of nuclear speckles, strongly contributing to the metastatic ability of cancer cells. Mou and colleagues have now reported that MALAT1 activity is not only controlled by epitranscriptomic modifications but also by complex RNA structures termed RNA G-quadruplexes (rG4) . rG4 takGenes (ISSN 2073-4425), “How Do New Genes Originate and Evolve?” ). This special issue belongs to the section “Population and Evolutionary Genetics and Genomics”. The interesting and multifaceted paper by Rubino et al [linc-UR-UB. There is beauty in the simplicity of formation of this gene; it involves a transcriptional read through from an existing gene to a small functional unit present in the downstream non-coding DNA region of the gene, thus creating a new gene and new function [linc-UR-UB transcript functions are fascinating: a lincRNA that may regulate the immune system by serving as a “sponge” for miRNAs, where the miRNAs themselves regulate expression of the human ubiquitin-specific peptidase 18 and thus regulate interferon [non-coding RNA Journal Club—9, abstract, “5. No Co-expression, No Sponging”, highlighted by Laura Poliseno [How new genes are formed is currently a major topic of interest, for example, see a special issue of  ISSN 207-4425, “H ISSN 207-4425, “H ISSN 207-4425, “HLiver is one of the most common distant metastasis sites and liver metastasis is one of the major causes of death among patients with colorectal cancer (CRC). Not surprisingly, the treatment and prevention of liver metastasis remains an urgent need that must be addressed in clinical practice of CRC. There are two major factors responsible for metastasis formation in CRC—the premetastatic niche and the tumor microenvironment. In a recent study, Sun et al. reported that hypoxic tumor microenvironment boosted the release of exosomes from cancer cells, comprising of miR-135a-5p and, more specifically, exosomal miR-135a-5p contributed to liver-specific premetastatic niche formation .In the study, the authors noted that the expression of both tumoral and serum miR-135b-5p was enriched in patients with CRC liver metastasis. Furthermore, their invitro and in vivo experiments revealed that CRC cells released miR-135a-5p enriched exosomes in hypoxic environment. Circulating miR-135a-5p enriched exosome could be phagocytosed by Kupffer cells, and exosomal miR-135a-5p initiated premetastatic niche formation in liver by promoting cell adhesion through LATS2-YAP-MMP7 axis. Exosomal miR-135a-5p also has immunosuppressive effect through CD30–TRAF2–NF-κB signal.These findings are truly promising for clinical practice of CRC as these suggest that inhibition of exosomal miR-135a-5p might serve as a potential target for treatment and prevention of CRC liver metastasis.Drosophila, mosquitos and mammals. TR has been identified in several eukaryotic organisms’ tissues with a predominance in neuronal cells. However, the precise molecular actors that control those TR events in a tissue specific manner remains mysterious. Karki et al. recently quantified TR of candidate genes in Drosophila melanogaster and characterized in detail the regulation of TR of the transcription factor Traffic jam (Tj) [Translational readthrough (TR) generates proteins with extended C-termini that can modify protein function. TR recoding occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR is utilized by viruses, yeast and higher eukaryotes such as jam (Tj) . They shNature [The article by M.E. Elguindy and J.T. Mendell on Nature , entitleNature Communications, describes a very interesting approach to study the substrate specificity of Dicer by combining a new RNA structure-probing method , together with an antibody pull-down of the enzyme [Dicer is a RNA endonuclease involved in the biogenesis of miRNAs and other regulatory non-coding RNAs. The characteristic structural shape of Dicer confers the property of acting as a “molecular ruler” that measures and cleave double-stranded RNAs (dsRNA) generating products with specific sizes. The manuscript by Luo and co-workers, published in e enzyme . The autThe transcriptional repressor Polycomb Repressive Complex 2 (PRC2) is a critical epigenetic regulator in various biological contexts. Beyond its chromatin binding capacity, PRC2 has attracted enormous interest for its interactions with RNA and many long non-coding RNAs (lncRNAs). Conversely, RNA can act as a catalytic inhibitor of PRC2 activity, although it has remained unclear how this inhibition is relieved in the contexts when lnRNA is present near chromatin region, without prior H3K27me3 being deposited.The study by Balas et al. describes a model for HOTAIR lncRNA and the RNA binding protein hnRNP B1 engaging in multivalent protein–RNA interactions. The inhibitory effects of HOTAIR on the PRC2 catalytic activity can be overcome by specific intermolecular RNA–RNA interactions of HOTAIR with its targets . There aSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease (COVID-19), which is of great health concern worldwide.Proceedings of the National Academia of Sciences, Joan Steitz’s laboratory investigated the impact of SARS-CoV-2 infection on host microRNA populations in human lung-derived cell lines and in nasopharyngeal swabs from SARS-CoV-2 infected individuals [In a recent paper published in ividuals . While nThe study indicates that CoV2-miR-O7a could contribute to SARS-CoV-2 pathogenesis. It aligns with a related study from the Cecere lab that confirms the existence of this viral miRNA-like molecules, possibly regulating host genes to evade interferon-mediated immune response .The Journal of Clinical Investigation, the authors characterize an ORF within the LINC00467 sequence, encoding for a micropeptide named ASAP (ATP synthase-associated peptide) for its ability to interact with ATP synthase subunits α and γ (ATP5A and ATP5C) [Long non-coding RNAs (lncRNAs) represent the greatest part of the human transcriptome, mainly acting as microRNA sponges, transcriptional scaffolds or epigenetic modulators; only recently, it has become apparent that open reading frames (ORFs) can be found even within lncRNAs transcripts. In a recent paper published on d ATP5C) . By eleg