Leviviridae  infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of  The advancement in sequencing, cultivation, and microscopy reveals that the phages are the simplest and the most abundant biological entities in the biosphere, with an estimate of 10ven time ,5. NeverPseudomonas aeruginosa (PA), which is sporadically found in serious nosocomial MDR bacterial infections and genuinely notorious for its high morbidity in immunocompromised patients suffering from cystic fibrosis or severe burns [Leviviridae phage , PP7 has one positive-sense, single-stranded RNA genome within an icosahedral capsid, which is 3,588 nucleotides in length and contains four genes encoding maturation protein (MP), capsid protein (CP), lysis protein (LP), and RNA replicase (RP). We have optimized the protocol based on a T7 promoter-driven transcription of the PP7 complementary DNA (cDNA) that is cloned into a mini-Tn7 vector for high-efficiency integration into the genome of a non-susceptible surrogate host strain, PAK.To further our understanding about the RNA phages, we describe the optimized method to create a reverse genetic system for the small RNA phage, PP7. PP7 is able to infect one of the multidrug-resistant (MDR) bacterial pathogen, re burns ,7. As a This protocol has been optimized to rapidly create a reverse genetic system for leviphages, which have small positive-sense single stranded RNA genomes of about 4000 nucleotides. The length is appropriate for an ordinary PCR reaction, obviating additional steps required for DNA assembly. The top strand  needs to be transcribed into RNA that should be fully functional as an mRNA for phage protein synthesis. Therefore, the first step of this protocol involves the extraction of genomic RNA from the phage particles followed by cDNA synthesis by reverse transcription-PCR (RT-PCR).Various steps of this protocol are schematically depicted in E. coli HB101 and pUC18T-mini-Tn7T-Gm (4569 bp) (GenBank accession number. AY599232) [glmS-PA5548 intergenic region of PAO1.Once the cDNA fragments are obtained, they need to be cloned for isolation or separation of single cDNA molecule, because the cDNA sequences are deemed varied due to the high mutation rate of the phage RP . BesidesY599232) . This coP. aeruginosa strains such as PAK and PA14, involving the two helper cells, i.e., the mobilizer cells and the transposase (pTNS2) donor cells [P. aeruginosa clones are then tested for their ability to produce plaques, as assessed by spotting or plaquing assay using the susceptible strains such as PAO1 and PMM49 [The HB101 transformants should be tested for their ability to generate functional phage particles. The plasmids from the selected HB101 clones need to be sequence-verified and then conjugation-mediated tetraparental mating is performed to deliver the cloned cDNA into the surrogate or cells . The selRNase free water TRIZOL Sodium chloride Potassium chloride Calcium chloride dihydrate Magnesium chloride hexahydrate Magnesium sulfate heptahydrate Tris-HCl, pH 7.5 Ethanol Chloroform Sucrose RNase-free DNase I set RNeasy MinElute clean-up kit Exprep Plasmid SV mini kit Superiorscript III Reverse Transcriptase 5× First-Strand buffer dNTP mixture (10 mM) 0.1 M DTT RNase inhibitor Phusion, High Fidelity DNA polymerase 5× High Fidelity buffer dNTP mixture (2.5 mM) Dimethyl sulfoxide SpeI HindIII 2.1 10× buffer T4 ligase T4 ligase 10× buffer Expin PCR SV Expin Gel SV Terminal deoxynucleotidyl transferase 5× A-tailing buffer dATP (2 mM) 4·7H2O)Phage buffer , 0.01 M MgSO2, 0.25 M MgCl2)5× KCM buffer Yeast extract Agar Cetrimide agar Agarose Gentamicin sulfate Carbenicillin disodium LB broth: 1% tryptone, 0.5% yeast extract, 1% NaClLB plate: 1% tryptone, 0.5% yeast extract, 1% NaCl, 2% Bacto-agarPseudomonas isolation): 4.53% cetrimide agar (Difco), 1% glycerolCetrimide agar (CA) plate Thermal cycler Incubator Shaking Incubator (Hanbaek Scientific)Heating block Eppendorf tubes Centrifuge (Eppendorf)Spectrophotometer MicroPulser Carry out all procedures at room temperature unless otherwise indicated.NOTE: Before starting the experiment, wash the benchtop with 70% ethanol to keep the environment RNase-free.11 pfu of PP7 to 100 μL using phage buffer in a 1.5 mL Eppendorf tube.Adjust the phage lysate containing ~1011 pfu of PP7 corresponds to 194.9 ng genomic RNA.NOTE: Based on the molecular weight of PP7 genomic RNA (1173 kDa), 102.Add 1 mL TRIZOL reagent and mix by pipetting.NOTE: TRIZOL reagent should be equilibrated at 4 °C before use.3.Incubate the mixture at room temperature for 5 min.4.Add 200 μL of chloroform, vortex vigorously for 15 s.5.Incubate the mixture at room temperature for 3 min.6.gCentrifuge the mixture at 4 °C for 15 min at 12,000× NOTE: Centrifugation results in the separation of the lower (pink) organic phase and the upper (colorless) aqueous phase. RNA remains exclusively in the aqueous phase).7.Transfer 300 μL of the aqueous phase to a fresh 1.5 mL Eppendorf tube.8.Add 750 μL of 100% ethanol to precipitate the RNA9.Incubate the mixture at −20 °C for for 1 h10.g and discard the supernatantCentrifuge the mixture at 4 °C for 10 min at 12,000× 11.Wash the RNA pellet twice with 1 mL of 70% ethanol12.g and discard the supernatantCentrifuge the mixture at 4 °C for 5 min at 7500× 13.Remove the supernatant and dry the RNA pellet (air-dry) for 5 min.NOTE: It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility.14.Dissolve the RNA with RNase-free water (20 μL) and incubate for 10 min at 55–60 °C in a heating block.NOTE: Incubation in a heating block facilitates RNA dissolution and unfolding.Adjust the RNA solution (from 3.1) to a volume of 85 μL with RNase-free water in a 1.5 mL Eppendorf tube.Add 10 μL RDD buffer and 5 μL DNase I from RNase-free DNase set (Qiagen) to the RNA sample .NOTE: DNase I solution should be stored at −20 °C to preserve its enzymatic activity.3.Incubate the RNA sample at room temperature for 20 min.4.Clean up the RNA sample with RNeasy MinElute Cleanup kit (Qiagen), according to the manufacturer’s instruction.NOTE: RNeasy MinElute Cleanup kit gave good results.5.Transfer the DNase-treated RNA sample (less than 5 μg) to an 0.2 mL thin-wall PCR tube and incubate in a thermal cycler at 65 °C for 5 min.6.Template RNA diluted in RNase-free water10.5 μL5× First-Strand buffer 4 μLdNTP mixture (10 mM) 1 μL0.1M DTT 2 μLRNase inhibitor (40 units/μL) 0.5 μLRT-Primer (PP7-3588R-H) (10 μM)1 μLSuperiorScript III Reverse Transcriptase (200 units/μL) 1 μLSet up the RT (reverse transcription) mixture in 0.2 mL thin-wall PCR tube as follows :7.Incubate the mixture in a thermal cycler at the following cycle setting:50 °C for 45 min → 70 °C for 15 min → 4 °CNOTE: This 3-step temperature setting is recommended for SuperiorScript III RT for cDNA synthesis.NOTE: Step 1  can be varied to increase the specificity and yield by raising the temperate (up to 55 °C) or increasing the time (up to 60 min), respectively.8.Template cDNA  diluted in water28 μL5× High Fidelity buffer 10 μLdNTP mixture (2.5 mM) 4 μLDMSO 2.5 μLForward primer (T7PP7-F-S) (10 μM) 2.5 μLReverse primer: RT-Primer (PP7-3588R-H) (10 μM)2.5 μLPhusion, High Fidelity DNA Polymerase (2 units/μL) 0.5 μLSet up the PCR mixture in 0.2 mL thin-wall PCR tube as follows :9.Hot start: 98 °C for 30 sAmplification (30 cycles): 98 °C for 10 s → 60 °C for 30 s → 72 °C for 2 minFinal extension: 72 °C for 5 min → 4 °CIncubate the mixture in a thermal cycler at the following cycle setting:10.Perform 0.8% agarose gel electrophoresis using the PCR sample.11.Perform cDNA gel extraction using the Expin Gel SV kit.Insert cDNA  or vector DNA diluted in water85 μL10× Digestion buffer (2.1 NEB)10 μLRestriction enzyme, SpeI (10 units/μL Enzynomics) 2.5 μLRestriction enzyme, HindIII (20 units/μL NEB) 2.5 μLSet up the digestion mixture in 1.5 mL Eppendorf tube as follows :Incubate the digestion mixture at 37 °C for 3 h.Clean up the digested DNA samples (either insert or vector) using the Expin PCR SV kit according to the manufacturer’s instruction.Insert (60–90 ng) and vector (20–30 ng) DNA diluted in water8 μL10× T4 DNA ligase buffer1 μLT4 DNA ligase 1 μL Set up the ligation mixture in a 1.5-mL Eppendorf tube as follows :NOTE: The DNA amount/length value should be three times for the insert over the vector.5.Incubate the ligation mixture at room temperature for 1 h.2 method [NOTE: Ligation mixture is subjected to either electroporation or chemical transformation for cloning. We generally use chemical transformation using a modified CaCl2 method .6.Add 20 μL 5× KCM buffer and 70 μL TDW to the ligation mixture.7.E. coli HB101) to the ligation mixture and gently mix.Add 100 μL competent cell (8.Place the mixture on ice for 10 min.9.Place the mixture at the heat block at 42 °C for 1.5 min10.Place the mixture on ice for 2 min.11.Add 1 mL LB to the mixture and incubate at 30 °C for 1 h in a shaking incubator.12.g for 5 min and discard supernatant.Centrifuge the mixture at 8000× 13.Resuspend the cell pellet with 200 μL LB broth and spread on LB agar plate containing gentamicin (25 μg/mL).14.Incubate the LB plate at 30 °C for 24–36 h.NOTE: The plate should be incubated at 28–30 °C rather than at 37 °C, to ensure slower growth.15.Pick the single colonies for isolation of the stable transformants, grow them in LB broth, and make the glycerol (20%) stock for storage.P. aeruginosa strain, PAO1. Refer to 3.5 Phage Production.NOTE: The correct transformants (designated HB101(PP7) hereafter) should be isolated for their ability to produce functional phage plaques on the PP7-susceptible E. coli strain, the pJN105-derivative plasmid containing the T7 polymerase gene (pJN-T7pol) (E. coli BL21(DE3)pLysS or into P. aeruginosa PAK. In this optimized protocol, the cloned cDNA is introduced into P. aeruginosa PAK.NOTE: To generate the surrogate N-T7pol)  can be iP. aeruginosa) and incubate the plates overnight at 37 °C (except for the HB101(PP7) that should be grown at 30 °C) to obtain fresh colonies.Streak frozen glycerol stocks of the parental bacteria onto LB plates or CA plates (only for E. coli HB101 containing pRK2013), the transposase-donor (E. coli SM10(λpir) containing pTNS2), and the surrogate recipient (P. aeruginosa PAK).NOTE: We have optimized the conjugation protocols using freshly grown cells. Bacteria used for tetraparental mating include the PP7-donor (HB101(PP7)), the PP7-driver .7.g for 5 min and resuspend with 20 μL of LB broth.Centrifuge the cell mixture at 8000× 8.Spread the resuspended cell mixture onto LB plate and incubate at 30 °C for overnight.NOTE: This tetraparental mating for multiple conjugation should be done at 30 °C to reduce the toxicity of the PP7 cDNA-containing cells. Therefore, the mating should be performed at least for 6 h.9.Scrape the cells from the LB plate with a scraper and resuspend in 200 μL of LB broth.10.Spread the cells onto the CA plate containing gentamicin (50 μg/mL).11.Incubate the CA plate at 30 °C for 24–36 h.12.Pick the single exconjugant colonies for isolation of the stable exconjugants and make the glycerol (20%) stock for storage.P. aeruginosa strain, PAO1. Refer to 3.5 Phage Production.NOTE: The correct exconjugants (designated PAK(PP7) hereafter) should be isolated for their ability to produce functional phage plaques on the PP7-susceptible NOTE: The pJN105 derivative plasmid expressing T7 polymerase (pJN-T7pol) can be introduced to PAK(PP7), only after the gentamicin marker has been removed by Flp-mediated excision as described at Steps 13 to 26 [13.Inoculate culture tubes containing 3 mL broth with fresh colonies of the stable exconjugants and incubate the tubes overnight in a shaking incubator.14.600 reaches 0.8.Inoculate culture tubes containing 3 mL LB broth by 1/100 diluting the seed culture and incubate the tubes until the OD15.g for 10 min and discard the supernatant.Centrifuge 3 mL of culture aliquot at 8000× 16.Wash the cell pellets with 1 mL of 10% sucrose solution.17.Resuspend the cell pellets with 200 μL of 10% sucrose solution in a 1.5 mL Eppendorf tube.18.Electroporate the cell suspension with 50 ng of pFLP2 DNA using MicroPulser.19.Incubate the cells at 30 °C for 1 h in shaking incubator.20.Spread the cells onto the LB plate containing carbenicillin (200 μg/mL).21.Incubate LB plate at 30 °C for 24–36 hNOTE: This step allows Flp-mediated marker excision.22.Check single colonies for antibiotic susceptibility by replicating each onto LB with gentamicin (50 μg/mL) and LB with carbenicillin (200 μg/mL).23.Incubate 30 °C for overnight or until the colonies appear.24.Streak carbenicillin-resistant but gentamicin-sensitive colonies onto an LB plate containing 5% sucrose.25.Incubate overnight at 30 °C or until sucrose-resistant colonies appear.sacB point mutation.NOTE: This step is to cure pFLP2. Almost all the sucrose-resistant colonies will have been cured of pFLP2, although a small fraction of colonies still have pFLP2 due to 26.Isolate the appropriate  stable single colonies and make glycerol (20%) stocks for storage.P. aeruginosa strain, the pJN105-derivative plasmid containing the T7 polymerase gene (pJN-T7pol) can be introduced into this unmarked PAK(PP7) strain. However, we have observed a severe growth defect of this unmarked strain by pJN-T7pol, which needs to be further characterized. We have used the strain from Step 12 for further analyses. This strain is able to form plaques on PAO1, even without the pJN-T7pol plasmid, which is most likely due to readthrough transcripts from the upstream promoters.NOTE: To generate the surrogate P. aeruginosa PAO1 is prepared in parallel except for incubation at 37 °C.Streak frozen glycerol stocks of the PP7-producing bacteria onto LB plates and incubate the plates overnight at 30 °C to obtain fresh colonies. Likewise, the PP7-suceptible NOTE: The procedure to test the bacterial colonies for their ability to produce functional phage particles is to streak from the plates onto the PAO1 lawn, as shown in 2.Inoculate culture tubes containing 3 mL LB broth with fresh single colonies and incubate the tubes overnight in a shaking incubator.NOTE: For large-scale production of functional phage particles from the phage producing cells, this overnight culture is used as the seed culture to inoculate multiples of LB broth (100 mL) in a 500 mL baffled flask.3.3.Transfer the PP7-producer culture to a 1.5 mL Eppendorf tube and add 0.1% CHCl3 permeabilizes the cell membrane to help release the functional particles inside of the cells. We do not use DNase I, which did not significantly improve the phage titer. In some cases, the phage titer was reduced by DNase I treatment, presumably due to the RNase contamination in some DNase batches.NOTE: CHCl4.g for 10 min and obtain the culture supernatant for the phage sample.Centrifuge the cultures at 12,000× 5.For phage titration, the phage sample is serially diluted in phage buffer.NOTE: The diluted RNA phage samples rapidly lose the infectivity even at 4 °C. Therefore, the phage titer measurement should be performed within 6 h after the serial dilution of the phage samples.6.Add the overnight-grown PAO1 cells (50 μL) to the 3 mL top agar and vortex briefly.7.Pour the top agar onto the pre-equilibrated LB plate and store 30 min at the clean bench.8.Spot the serially diluted phage samples on the LB plate.9.Incubate the LB plate at 37 °C for overnight (12–18 h).NOTE: This spotting assay is for rough estimation of the phage titer. For precise enumeration of the phage titers, the standard protocol for phage plaque assay should be performed on a clean bench. For this, the phage samples appropriately diluted based on the spotting assay results are subjected to binding to the PAO1 cells for 15 min. This phage-cell mixture can be applied to the top agar, as in Step 6.NOTE: The PP7 phages produced from the cDNA-containing clones can be verified by deep-sequencing to compare the quasi-species distribution between those produced from the normal PP7 infection cycles. In this protocol to verify the genomic structure of the produced phages, we exploited 5′-RACE (rapid amplification of cDNA-ends), in that the 5′-end of the T7 promoter-driven transcripts differs from that of the PP7 genome.Perform the steps of 3.1 RNA Extraction using the phage samples obtained from 3.5. Phage Production.NOTE: Small-scale RNA extraction can be done for 5′-RACE using the single plaques after spotting assay of 3.5 Phage Production.2.Perform the steps 1 to 5 of 3.2 cDNA Synthesis.3.Template RNA diluted in RNase-free water10.5 μL5× First-Strand buffer 4 μLdNTP mixture (10 mM) 1 μL0.1M DTT 2 μLRNase inhibitor (40 units/μL) 0.5 μLRT-Primer (PP7-340R) (10 μM)1 μLSuperiorScript III Reverse Transcriptase (200 units/μL) 1 μLSet up the RT mixture in 0.2 mL thin-wall PCR tube as follows :4.Incubate the mixture in a thermal cycler at the following cycle setting:50 °C for 45 min → 70 °C for 15 min → 4 °CNOTE: This temperature setting is recommended for SuperiorScript III RT for cDNA synthesis.5.Perform cDNA extraction using the Expin PCR SV kit.6.Template cDNA  (200 ng) in water13.5 μL5× A-tailing buffer 4 μLdATP (2 mM) 1 μLTerminal deoxynucleotidyl transferase (20 units/μL) 1.5 μLSet up the A-tailing mixture in 0.2 mL thin-wall PCR tube as follows :7.Incubate the mixture at 37 °C for 30 min and then at 70 °C for 10 min.8.Template cDNA  diluted in water28 μL5× High Fidelity buffer 10 μLdNTP mixture (2.5 mM) 4 μLDMSO 2.5 μLForward primer (PP7-319R) (10 μM) 2.5 μLReverse primer (dT adapter) (10 μM)2.5 μLPhusion, High Fidelity DNA Polymerase (2 units/μL) 0.5 μLSet up the 5′-RACE mixture in 0.2 mL thin-wall PCR tube as follows :9.Hot start: 98 °C for 30 sAmplification (30 cycles): 98 °C for 10 s → 60 °C for 30 s → 72 °C for 2 minFinal extension: 72 °C for 5 min → 4 °CIncubate the mixture in a thermal cycler at the following cycle setting:10.Perform 0.8% agarose gel electrophoresis using the 5′-RACE product.11.Perform DNA extraction using the Expin Gel SV kit for sequence verification.P. aeruginosa leviphage, PP7. Most genetic manipulation systems (T7 polymerase-promoter system and plasmid-associated features) employed in this study are stemmed from E. coli rather than P. aeruginosa. While they are all supposed to work in other bacteria, the efficiency needs to be optimized in that the genetic signature of P. aeruginosa clearly differ from that of E. coli pLys) shows ~103 fold increase in phage production compared to the E. coli HB101 clone. As shown in While we can identify the functional phage assembly from the pression A. This sAs the original design was based on the T7 polymerase-promoter system, it is inevitable to include additional 3 G’s at the 5′-end of the PP7 cDNA. Due to this, we need to identify the 5′-end of the RNA genome of the PP7 virion derived from the PP7 cDNA. To this end, 5′-RACE experiments were performed using the phage plaques obtained from This protocol could be also exploited without modification to create a reverse genetic system for almost all leviphages. Using this protocol, we have also created the reverse genetic system for the best-characterized coliphage, MS2 as well . The cDN


The effect of (Tb+Y)/Al ratio on microstructure evolution and densification process was investigated in detailed. The results showed that the grain growth kinetics were significantly affected by (Tb+Y)/Al ratio. Al-rich and Tb-rich phases appeared in part of the samples of different ratios. Particularly, excess aluminum increased the diffusing process, leading to a higher densification rate, while samples with excess terbium ratios displayed a smaller grain size and lower relative density. The optical quality was highly related to the amount of the secondary phase produced by different (Tb+Y)/Al ratios. Finally, (Tb0.6Y0.4)3Al5O12 transparent ceramics have been fabricated through pre-sintering in vacuum, followed by hot isostatic sintering (HIP), and the best transmittance of sample with a 4 mm thickness was approximately 78% at 1064 nm.(Tb At solators ,5,6. Comications . Howeverications ,9,10. Alications ,12. This1−xRx)3Al5O12  ceramics by a two-step sintering method, and confirmed that Y3+ addition improved the optical quality of the TAG ceramics. Duan et al. [1−xYx)3Al5O12 transparent ceramics with ultralow optical loss for practical applications, promoting the commercial development of TAG transparent ceramics.TAG transparent ceramic has been studied for many years since it was firstly reported in 2011 . A largen et al.  found Y-n et al.  produced2O3-Al2O3. Much research has been devoted to understanding the effect of composition deviation on several common garnet structures. Hu et al. [2O3 pinned in the grain boundary limited the fast migration of grain boundaries in Pr: LuAG transparent ceramics. Stanek et al. [2O3 and Y2O3. However, related works have not been carried out in TAG ceramic system, though it would be meaningful for obtaining transparent ceramics with excellent magneto-optical properties.Even though most former studies have claimed that highly transparent TAG or TAG-based ceramics were fabricated, there was still numerous scatters that existed in the samples  which limited further improvement of the transmittance. Generally, in order to avoid the second phase, the ratio of RE/Al  must be carefully controlled as 3/5, according to the binary phase diagram of REu et al.  found thk et al.  studied k et al.  investig4O7 instead of Tb2O3 is usually used as the raw material to prepare TAG transparent ceramics, owning to the instability of Tb2O3 at room temperature [3+ and Tb4+ in Tb4O7 powder can be hardly measured. In other words, Tb4O7 should actually be described as Tb4O7±x, making precise control of Tb/Al ratios impossible. Therefore, investigate different ratios will be significant for fabrication of high optical quality ceramics. In this paper, (Tb0.6Y0.4)3Al5O12 transparent ceramics were fabricated by reactive sintering in vacuum, followed by hot isostatic sintering (HIP) treatment and Tb4O7, Y2O3, and α-Al2O3 were used as raw powders. The effect of (Tb+Y)/Al ratio on the phase formation, densification process, and microstructure evolution was elaborately investigated.Generally, Tbperature ,21. Howe0.6Y0.4)3Al5O12 transparent ceramics with different (Tb+Y)/Al mole ratios  were fabricated with high-purity commercial Tb4O7 , Y2O3 , and α-Al2O3  powders. The particle sizes of three raw powders were 2, 1, and 0.2 μm, respectively. Meanwhile, 0.5 wt % TEOS  and 0.1 wt % MgO  were added as sintering aids. The powder mixtures was dispersed in 99.99% ethyl alcohol and ball-milled in nylon tank for 15 h. Then, the slurries were dried at 80 °C in oven for 24 h and sieved through a 100-mesh screen. After this, they were uniaxially pressed into plates in Φ 12 mm stainless steel molds at 20 MPa and cold isostatic pressing at 200 MPa for 5 min. In order to remove organics, plates were calcined at 800 °C for 6 h in a muffle furnace. The green bodies were pre-sintered at varieties temperatures (from 950 to 1550 °C) in a vacuum furnace  under a vacuum level of ~10−3 Pa for 4 h, followed by HIP at 1600 °C under 196 MPa Ar atmosphere. Finally, all samples were annealed at 1350 °C for 10 h in a muffle furnace , and mirror-polished to 4 mm on double sides. with Cu Kα radiation. The microstructures of the ceramics were characterized by scanning electron microscopy . The element mapping was conducted with energy dispersive spectroscopy . The densities of the transparent ceramics were measured by Archimedes method. The in-line transmittances of the polished samples were measured by UV–vis–NIR spectrophotometer .2O3 and form a solid solution of yttrium terbium aluminum garnet (YTbAG). Specifically, Tb4O7 deoxygenates to Tb2O3 beyond 950 °C and Tb2O3, Y2O3, and Al2O3 can be detected at this temperature. Yttrium terbium aluminum monoclinic phase (YTbAM) forms at 1050 °C while the diffractions of raw powders still exist. With the temperature increasing, YTbAM and yttrium terbium aluminum perovskite phase (YTbAP) simultaneously appear at 1150 °C, and the diffraction intensity of YTbAM decreases. Meanwhile, YTbAP and some YTbAG are detected at 1250 °C while YTbAM has disappeared. When the temperature reaches 1350 °C, a pure YTbAG phase is generated and all peaks match well with TAG standard card (PDF#17-0735). No residual intermediate phases remain to be detected. In summary, YTbAM, YTbAP, and YTbAG appear in order with the reaction processing, which can be described by the formulasThe X-ray diffraction patterns in The relationship between relative density and pre-sintering temperature is shown in 2O3 second phases are detected in the sample with a ratio of 0.6000. Due to the lower atomic number, it looks darker. Meanwhile, Tb and Y disappear in this area. The mapping result of 0.6073 ratio is showed in SEM micrograph and the corresponding EDS mapping images of various elements of the pre-sintered sample with two (Tb+Y)/Al ratios (0.6000 and 0.6073) are shown in The rare earth-controlled densification and average grain size were already discovered in YAG ,25. In tHIP treatment is a typical method in fabricating optical ceramics, because it can remove residual intergranular pores and further improve the densification. There is more suitable microstructure of pre-sintering ceramics before HIP due to no intragranular pores, higher relative density, and smaller grain size. Therefore, the microstructures of samples pre-sintered at 1450 °C and treated by 1600 °C HIP are displayed in 3+: 7F6 → 5D4 transition. The best optical quality sample, whose transmittance reaches 78% at 1064 nm, is obtained by a (Tb+Y)/Al ratio of 0.6036. Macroscopic observations of (Tb0.6Y0.4)3Al5O12 transparent ceramics with different (Tb+Y)/Al ratios are displayed inside. The samples with 0.6036, 0.6073, and 0.6110 ratios are transparent, with words being clearly seen below the photograph, while 0.5964 and 0.6000 ratio samples are opaque. The bright yellow appearance is connected with the valence state of terbium.0.6Y0.4)3Al5O12 transparent ceramics were fabricated by vacuum pre-sintering and HIP treatment. Due to the uncertain volume of terbium in Tb4O7 raw powders, the influence of different (Tb+Y)/Al ratios on the densification process and optical properties of (Tb0.6Y0.4)3Al5O12 transparent ceramics were studied in detail. Meanwhile, results indicated that excess aluminum ((Tb+Y)/Al = 0.5964 and 0.6000) made the densification process faster, while excess terbium ((Tb+Y)/Al = 0.6073 and 0.6110) caused decay and hindered grain growth. More importantly, the excess aluminum or terbium caused second phases to appear, which seriously affected the sample optical properties. Finally, transparent ceramic with (Tb+Y)/Al = 0.6036 pre-sintered at 1450 °C in vacuum, followed by HIP treatment at 1600 °C, resulted in better optical quality and a transmittance of up to 78% at 1064 nm in a sample of 4 mm thickness was obtained.(Tb


Exopalaemon carinicauda by using ion-exchange resin chromatography (IEC) and gel filtration. These two chitinases, named EcChi1 and EcChi2, were identified by chitinolytic activity assay and LC-ESI-MS/MS. Their apparent molecular weights were 44 kDa and 65 kDa as determined by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of EcChi1 and EcChi2 was 1305.97 U·mg−1 and 28.69 U·mg−1. The optimal temperature and pH of EcChi1 were 37 °C and pH 4.0, respectively. Co2+, Fe3+, Zn2+, Cd2+, and Cu2+ had an obvious promoting effect upon chitinase activity of EcChi1. For colloidal chitin, the Km and Vmax values of EcChi1 were 2.09 mg·mL−1 and 31.15 U·mL−1·h−1.In this paper, we purified two native chitinases from the hepatopancreas of the ridgetail white prawn  Chitin is the second most abundant organic compound on Earth. It is widely spread in various organisms and almost 10% of the global landings of aquatic products consist of organisms rich in chitinous materials  since itO-glycoside hydrolase superfamily, have shown immense potential for hydrolyzing chitin in the most economical way. Specific combinations of chitinolytic enzymes are necessary to obtain the desired chain length of the resulting oligomer [N-acetyl-glucosaminidase and exochitinase activity, whereas the production of GlcNAc requires a higher proportion of exochitinase and N-acetyl-glucosaminidase activity [Chitinases EC3.2.1.14), a member of the oligomer . For exaactivity ,9. In ad.14, a meactivity .Uca pugilator [Marsupenaeus japonicas [Penaeus monodon [Fenneropenaeus chinensis [Litopenaeus vannamei [Pandalopsis japonica [E. carinicauda. The enzymatic characteristics and kinetic parameters of the chitinase with higher activity (EcChi1) were studied.Abundant research related to chitinases has been done in microorganisms, plants and insects ,14,15,16ugilator , Marsupeaponicas ,25,26, P monodon ,28,29, Fhinensis , Litopenvannamei  and Pandjaponica  have beeE. carinicauda by anion-exchange chromatography and gel-filtration chromatography (E. carinicauda transcriptome (ECT) database, the purified EcChi1 and EcChi2 with chitinolytic activity were definitely chitinases. Based on ECT data and SMART RACE method, we obtained the full-length cDNA sequences of EcChi1 and EcChi2. The full-length nucleotide sequences and the deduced amino acid sequences are shown in E. carinicauda by cation exchange resin (HiTrap CM FF).Two proteins with chitinolytic activity, named EcChi1 and EcChi2, were purified from hepatopancreas of tography . Accorditography . Based o−1 and 8.28 U·mL−1. The protein concentration of EcChi1 and EcChi2 was 0.0167 mg·mL−1 and 0.289 mg·mL−1, respectively. The specific activity of EcChi1 and EcChi2 was 1305.97 U·mg−1 and 28.69 U·mg−1, respectively.The chitinolytic activity of EcChi1 and EcChi2 was 21.80 U·mLP. japonicas chitinase purified from the liver [L. vannamei chitinase purified from the digestive gland [The results above showed that the purity and amount of EcChi1 was much higher than that of EcChi2. Hence, the purified EcChi1 was selected to study its enzymatic characteristics, including the optimal pH and temperature, effects of metal ions and enzyme stabilities. The results showed that EcChi1 had maximal chitinolytic activity at pH 4.0 a and washe liver  and L. vve gland  had an oMeganyctiphanes norvegica [E. superba and M. norvegica, which were 50% inactivated after 60 min incubation at 46 °C [To determine the optimal temperature and thermal stability of EcChi1, the chitinolytic activity at different temperatures (from 20 to 60 °C) was measured. The results indicated that the apparent optimal temperature of EcChi1 was 37 °C b which worvegica . The enzat 46 °C .2+, Fe3+, Zn2+, Cd2+ and Cu2+ had an obvious promoting effect upon chitinase activity of EcChi1 when incubated with enzyme for 20 h. Compared to the control, the chitinase activity of EcChi1 with these ions were increased 300%–400%. Besides, Fe2+ also had a certain positive effect on EcChi1 activity. The effect of other ions on EcChi1 activity was similar to that of the control , Gel filtration resins (HiPrep 16/60 Sephacryl S-100 High Resolution), HiPrep Desalting resins  were purchased from GE Healthcare . Chitin (from shrimp shells) was purchased from Sigma , and colloidal chitin was prepared using the reported method [N-Acetyl-D-glucosamine was purchased from Life Science .The ridgetail white prawns, d method . 4- and 50 μL enzyme solution. After digestion for 2 h at 37 °C, the reaction was terminated by centrifuged at 4000 rpm, for 10 min. 25 μL 1% (w/v) potassium tetraborate was added to 50 μL supernatant of the standard or samples, respectively. The tubes were heated in the boiling water bath for 7 min and cooled rapidly in cold water. Then 250 μL acetic acid and 125 μL DMAB were added to each tube and all tubes were placed in a water bath at 37 °C after mixing. Twenty minutes later, all tubes were cooled in cold water and the absorbance measured at 585 nm. Blank (control assay) was done with inactivated enzymes. One unit of chitinase activity was defined as the amount of enzyme, which releases 1 μg of N-acetyl-d-glucosamine per hour from colloid chitin under the conditions described above.The chitinolytic activity was measured by quantitative estimation of the reducing sugars released during the hydrolysis of colloid chitin via the method of Reissig et al. . N-Acety−1 BSA (2–12 μg) were used to draw standard calibration curve. The specific activity was expressed as units of activity per milligram of protein (U·mg−1 of protein).The protein concentrations were determined by the method of Bradford . VariousThe hepatopancreas were dissected from 100 ridgetail white prawns and incubated by Tris solution  for 1 h at 4 °C. The mixture was centrifuged at 10,000 rpm, 4 °C for 15 min and the precipitate was discarded. The pH of the supernatant which contained crude protein was adjusted to pH 7.0 with 1 M HCl.2HPO4-NaH2PO4 buffer . And it was desalted with HiPrep Desalting resins. During purification process, chitinase activity was assayed using the method described above.The isolation and purification of chitinases was performed in three steps. The supernatant was precipitated with ammonium sulfate at 70% saturation, and kept at 4 °C overnight. Then the precipitated proteins were collected by centrifugation at 10,000 rpm, 4 °C and resuspended by 30 mL Na2HPO4-NaH2PO4 buffer, and the adsorbed protein was eluted by linear gradient with sodium phosphate buffer containing 1 M NaCl. The fractions with high elution peak were collected and its chitinolytic activity measured. The high chitinase activity fractions was gathered, desalted and concentrated.The total protein containing chitinase was isolated by using anion exchange resin (HiTrap Q FF) and cation exchange resin (HiTrap CM FF) in sequence. The resins were equilibrated with 20 mM, pH7.2 Na−1. One milliliter per fraction was collected. The chitinase activity of each elution peak was determined.Then, the concentrated sample was subjected to a gel filtration resin (HiPrep 16/60 Sephacryl S-100 High Resolution). During the process, 50 mM, pH 7.2 sodium phosphate buffer contained 0.15 M NaCl was adopted, the amount of sample application was 2 mL, and the procedure was run at a flow rate of 1 mL·minThe molecular mass of each purified protein was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12% separating gel and 5% stacking gel. The gel was stained with Coomassie brilliant blue.E. carinicauda transcriptome (ECT) database [The bands with chitinolytic activity were excised from gel for protein identification via liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The Mascot software was used to analyze the mass spectrometric data combined with database .The optimal pH of the purified chitinase was determined by varying the pH of reaction mixture from 2.2 to 9.0. The buffer used to generate pH ranges were 50 mM glycine-HCl buffer (pH 2.2–3.0), sodium acetate buffer (pH 4.0–5.0), sodium phosphate buffer (pH 6.0–8.0), and sodium carbonate buffer (pH 9.0).The stability of chitinase activity at different pH was also determined. The purified chitinase was incubated with different pH buffers mentioned above at 37 °C for 24 h without substrate, respectively. After incubation, the residual activity of the treated enzyme was measured. The activity at the optimal pH without incubation was taken as 100%.The measurement of the optimum temperature was conducted by putting reaction mixtures in different temperatures (ranging from 20 to 60 °C) at the optimal pH condition and the amount of reducing sugars released from colloid chitin was measured.To determine the thermal stability, chitinase was incubated at different temperature (ranging from 20 to 70 °C) for 24 h. Then the residual activity was measured as described in 2+, Mg2+, Cu2+, Li+, Fe2+, Fe3+, K+, Cd2+, Co2+ and Zn2+) were used to identify their effect on chitinolytic activity. Each metal ion was added into reaction mixture with a final concentration at 10 mM, and then the chitinolytic activity was determined immediately following the method described above.Several metal ions  for 20 h at 4 °C. A control assay was done in the reaction without metal ions and its activity was taken as 100%.m and Vmax) of EcChi1, different concentrations of substrate  ranging from 0.2 to 10 mg·mL−1 was mixed with 50 μL enzyme in sodium acetate buffer , and incubated at 37 °C for 2 h. The activity was measured as described in m and Vmax values were determined by Lineweaver-Burk plot.To determine the kinetic constants (KE. carinicauda. SDS-PAGE gel showed that the apparent molecular weight were 44 kDa and 65 kDa, respectively. Some biochemical and enzymatic properties of EcChi1 were characterized. The optimal pH, optimal temperature, pH stability, and thermal stability of EcChi1 were pH 4.0, 37 °C, pH 5.0, and below 40 °C. The activities of EcChi1 were strongly activated by Co2+, Fe3+, Zn2+, Cd2+, and Cu2+. And EcChi1 exhibited Km values of 2.09 mg·mL−1 and Vmax values of 31.15 U·mL−1·h−1 when using colloidal chitin as substrate. These results will provide a certain basis for the application of EcChis in the future.In this study, two chitinases, EcChi1 and EcChi2, were isolated from hepatopancreas of


O-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5′ modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs ∼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5′ and 3′ linkers. Unlike mRNA, small RNA often contains modifications including 5′ cap or triphosphate and 2′- High-throughput sequencing has become a revolutionary technique for analyzing DNA/RNA. As the cost has significantly decreased over the past decade, it is becoming a standard tool for gene analyses in biomedical fields. Therefore, any technical advance in this technique will have a broad impact. Since a single sequencing run is usually sufficient for analyzing multiple samples, individual samples are usually cloned with specific barcodes, pooled, and sequenced as a single library for cost-sharing, and then debarcoded to obtain sample-specific sequences . AlthougUsually DNA, mRNA, and small RNA sequencing libraries are constructed with distinct linkers and/or chemistry, forcing customers to purchase different kits . Lack ofCaenorhabditis elegans 22G-RNA (22G) bears a 5′ triphosphate, which is incompatible with 5′ ligation . Unlike the previously reported methods, these treatments are coperformed in the linker ligation reactions, avoiding the steps for enzyme removal/inactivation and/or buffer exchange. All the steps are performed inside a PCR tube in an all-liquid-based manner, significantly reducing labor time. This method is able to construct a small RNA library using as little as ∼20 ng of total RNA, a level much lower than the amount required by available commercial kits. Since it is all-liquid-based, it can be adapted to automation. The cost is minimal since the method only needs a few common enzymes, which can be purchased or easily purified using a single His-tag purification. In all, our strategy is more sensitive, convenient, versatile and cost-effective than most available methods.We have developed a unified strategy for constructing high-throughput small RNA/mRNA libraries. Since the linkers are derived from a strategy for making high-throughput DNA libraries, the barcoded PCR primers here can be used to amplify high-throughput DNA libraries too. Our strategy is capable of cloning modified and unmodified small RNAs using the enzymatic treatments including dephophorylating RNA by 5′GCUCUUCCGAUCU-OHOH-ACACUCUUUCCCUACACGAC is an RNA oligo with 5′OH blocking the RNA ligase-mediated activation (adenylylation with ATP) (5′AGATCGGAAGAGCACACGTCTGAACTCCAGTCAddCApp- is an activated DNA oligo (adenylylated) which allows for ligation without ATP, that is, serving as a ligation donor with the 3′ ddC (dideoxyC) blocking its usage as a ligation acceptor  of the 5′ linker and the first 13 nt of the 3′ linker ligated to each target DNA strand form double-stranded DNA, a commonly used strategy required for DNA-based ligation . Since with ATP) . Therefoacceptor . This de linkers B.Supplemental Material .A complete list of the ligation linkers, RT primer, and PCR primers including 64 barcoded 3′ primers are provided in the Supplemental Table S2, small RNA is first ligated with 0.5 µM activated 3′ linker using 0.5 µM truncated T4 RNA ligase 2 in 10 µL buffer containing 50 mM Tris (pH 7.5), 10 mM DTT, and 10 mM MgCl2. Since the 3′ linker is 3′ ddC-modified primarily for blocking 3′ linker self-ligation, it can only be ligated to the 3′OH of target RNA. This strategy also avoids the activation of the 5′ phosphate of target RNA since (i) no ATP is used and (ii) the truncated T4 RNA ligase 2 does not possess the adenylylation activity  denaturing the truncated T4 RNA ligase 2; and (ii) annealing with ∼0.5 µM RT oligo to the 3′ linker either ligated or unligated by adding 0.5 µL of a 10 µM RT oligo. And then 0.4 µM 5′ linker, 0.5 mM ATP, 0.25 µM RNA ligase 1, and water are added to the reaction reaching a final volume of 20 µL for the 5′ ligation. T4 RNA ligase 1 activates the 3′-ligated target RNA if it contains or has been enzymatically treated to expose 5′ monophosphate (5′p). In contrast, the 5′ linker lacking 5′p cannot be activated. Therefore, the 5′ linker can only serve as a ligation acceptor ligated to the 3′-ligated and 5′-activated target RNA. The annealing of the RT oligo with the 3′ linker may reduce the ligation of the free 3′ linker, if any are left after the 3′ ligation, with the 5′ linker, since T4 RNA ligase 1 prefers single-stranded substrates. This strategy may significantly reduce the formation of 5′ linker-3′ linker ligation especially when the RNA substrate is much less than the 3′ linker, generating excessive 3′ linker, as discussed below in the single worm RNA cloning.A 30-minute RT step follows the 5′ ligation with addition of ∼0.2 µM Superscript II or III (Invitrogen), ∼4 mM additional DTT, ∼0.4 mM dNTP, and ∼2 µL 10× RT dilution buffer (0.25 M Tris pH 8.8 and 0.75 M KCl) reaching a final volume of ∼24 µL. Then the enzymes are inactivated at 85°C for 5 min. The obtained cDNA is amplified and extended to obtain the full-size linkers by PCR. 8-nt barcodes are inserted in the 3′ PCR primers for labeling individual samples. A typical 50 µL PCR reaction is composed of 1× PFU buffer, 15 mM tetramethylammonium chloride for reducing primer dimmer, 0.1 mM dNTPs, 0.1 µM 5′ and 3′ primers, 5 µL RT reaction, and 1× PFU polymerase. The PCR is first amplified for 5 cycles  and then amplified for 11 cycles . Additional 0.6 µM 5′ and 3′ primers are added and the PCR is amplified for 2 more cycles .Supplemental Table S2.In all, the whole cloning process is performed in a single PCR tube with liquid components added sequentially and can be easily finished within ∼7 h including ∼ 1-h labor time. A formulated working protocol is presented in the C. elegans adult worms, and using purified C. elegans small RNA of size less than 200 nt, which contain miRNA, 22G-RNA (siRNA), 21U-RNA (piRNA), tRNA, and 5.8S/5S rRNA  or other byproducts. The result clearly indicates that our method is able to clone small RNA at fmole level from single worms and deliver high reliability at this level, as the two individual worm samples exhibited consistent small RNA profiles D.C. elegans PIR-1 works at 20°C, it is a perfect candidate for the desired activity. We obtained a recombinant C. elegans PIR-1 of homogeneity from E. coli . To examine its dephosphorylation activity, we coapplied this enzyme in the 3′ ligation step with the truncated T4 RNA ligase 2, generating 3′ ligated and 5′ p-RNA using the same reaction. Then the RNA was further 5′ ligated. The high-throughput sequencing analysis confirmed that this strategy worked efficiently for cloning ppp-RNA. As shown in Small ppp-RNA cannot be directly ligated at the 5′ end. To clone it, ppp-RNA is usually treated with commercial RNA polyphosphatases, generating 5′ monophosphorylated RNA (p-RNA), which is compatible with 5′ ligation . HoweverE. coli and examined its decapping activity in the ligation buffer. hDcp2 worked well in the 5′ ligation step with T4 RNA ligase 1  or promoter-associated small RNA is generated during transcription initiation and bears a 5′ G cap with size <200 nt . The capligase 1 B, as theligase 1 B or circreported .In our condition, hDcp2 does not work well when added in the 3′ ligation step (data not shown). Since the buffer conditions for the 3′ and 5′ ligations are almost the same, we suspect that the hDcp2 we purified may be contaminated with a low level of RNA phosphatases, which dephosphorylated the p-RNA generated by hDcp2 in the 2-h 3′ ligation step, making it incompatible for the following 5′ ligation. In contrast, when hDcp2 is added in the 5′ ligation, the ligase may add a linker to the 5′p of the decapped RNA before dephosphorylation by any RNA phosphatase.In addition to cloning small RNA, we aimed to use the same method to clone mRNA. In general for mRNA cloning, mRNA is fragmented to small RNA, ligated with linkers, and converted to cDNA. However, a partial digestion of mRNA either chemically or enzymatically usually generates small RNA with 5′OH and 2′p or 3′p or cyclic phosphate at the 3′ end. For ligation-based cloning methods, these ends have to be converted to 5′p and 3′OH. To avoid these conversion steps, we utilized nuclease P1, an enzyme cutting single-stranded RNA or DNA, generating small RNA or DNA with 5′p and 3′OH . A potenSupplemental Fig. S2A) and the individual gene expression levels correlate well between the two samples . Moreover, our method was capable of cloning at least 1 ng mRNA (data not shown), a sensitivity level sufficient for most routine work.We then constructed a library with poly (A)-containing mRNA fragmented using nuclease P1. The obtained mRNA profile is similar to that of a previous one made using alkali hydrolysis. For example, the reads spread randomly along the length of mRNA  and BB is ∼0.01% (0.01%/[98%+0.01%]). Therefore, if perfectly matched products are selected, abundant DNA species are overrepresented, and if bulged products are selected, less abundant DNA species are overrepresented.To solve the overcycle issue, theoretically both perfectly matched and bulged products can be purified together. However, PCR products often contain primer dimers and undesirable products, which form bulged products with desirable PCR products once PCR reactions are overcycled, basically making this purifying-all strategy ineffective. To solve this issue, we used to examine the PCR product at cycle number 12, 16, 20, and 24 from the same PCR reaction on a PAGE gel, obtain the maximal PCR number without overcycle, and use this number to mass produce the product . AlthougA high-throughput sequencing library usually contains multiple barcoded samples for cost-sharing. To obtain a specific composition, individual samples are purified, quantified and then mixed as a pooled sample. Since samples may have primer dimers and/or other byproducts including cDNA derived from rRNA and tRNA, a gel purification is required for obtaining target cDNA of specific sizes. This process is tedious and time consuming . To reduOur major goal is to design a simple, convenient and cost-effective method for cloning small RNA. For this purpose, we have developed an all-liquid-based multistep reaction in a single PCR tube. The whole procedure takes ∼ 7 h with only ∼ 1 h labor time and the rest primarily used for increasing ligation efficiency of modified RNA. It can be shortened to ∼4 h for cloning unmodified miRNA . Like the commercial kits, our method starts with total RNA, a strategy providing convenience but generating byproducts derived from tRNA and rRNA. In addition, the whole procedure involves no gel purification or DNA precipitation . Our method is much more sensitive than the commercial kits, working with as little as 16 ng total RNA. We speculate that we could start with even less total RNA if using one gel purification to enrich target cDNA amplicons while removing primer dimmers and linker-linker ligation byproducts, followed by a second round of PCR amplification for mass producing target cDNA amplicons.We prefer ligation reactions for adding 5′ linkers to target RNA over other techniques, for example, cDNA circularization or template switching via reverse transcriptases. Since most degraded RNA bears a 5′OH, it cannot be cloned using ligation, which requires a 5′p. Therefore, not only does the 5′ ligation add a linker for cDNA application and sequencing, but also serves as a selection mechanism for enriching authentic small RNAs. Due to this selection, our method works well with samples heavily contaminated with degraded RNA including some immunoprecipitated RNA samples. At the same time, our method can be easily adapted to clone RNA bearing a 5′OH simply by adding T4 Polynucleotide Kinase (PNK) in the 5′ ligation step (data not shown).Our method only needs a few common enzymes such as T4 RNA ligases, reverses transcriptases, DNA polymerases and PIR-1 and hDcp2. The last two are required for processing 5′ modified RNA but not for p-RNA including miRNA, Dicer-dependent siRNA, and piRNA. We easily obtained RNase-free enzymes using a single His-tag-mediated purification. The first three enzymes above are also commercially available. However, for some reasons, when tested by other labs, the protocol did not work well with some commercial enzymes, but worked well with our purified enzymes. We believe that the failure may be caused by the commercial T4 RNA ligase since commercial SuperScript III and Taq worked in our method. We speculated that the commercial T4 RNA ligases used may be contaminated with a trace amount of RNases or phosphatases. We estimate that the cost per library construction is negligible if using home-made enzymes and activated 3′ linkers, and less than $10 if using commercial enzymes and 3′ activated linkers.O-methyl at the 3′ end, which decreases the 3′ ligation efficiency. This inhibitory effect is overcome by addition of 25% PEG-8000   . csRNA i22G-RNA) . To enri22G-RNA) . 5′ OH-RWe aimed to develop a versatile method for cloning both mRNA and small RNA and to use the same sets of PCR primers to amplify libraries derived from DNA and RNA. Since the 5′ and 3′ linkers are derived from the DNA cloning system, the PCR primers definitely work with DNA libraries. Actually, we used these primers to amplify DNA libraries constructed using a commercial kit (data not shown) without purchasing the expensive barcoded primers. For mRNA cloning, we first used alkali-hydrolysis to fragment mRNA, generating small RNA with 5′OH and 3′ cyclic phosphate. Then we used PNK to fix the ends in the 3′ ligation step by: (i) removing the 3′ cyclic phosphate at room temperature for ∼5 h; and (ii) phosphorylating the 5′ end with addition of ATP at 37°C for 1 h (data not shown). Although it works well, the 3′ ligation step takes ∼6 h, making it impossible to finish the cloning process within 1 d. This prompts us to use nuclease P1, which generates ligation-compatible ends. The cost of nuclease P1 is close to alkali-hydrolysis, basically nothing. Nuclease P1 works well at 60°C, significantly reducing the effect of RNA secondary structures on the digestion pattern and efficiency. Moreover, our partial digestion condition is insensitive to the RNA amount so that there is no need to optimize the enzyme/RNA ratio for getting fragmented RNA of desired size.All in all, our method is convenient, sensitive, and versatile. Any laboratory with basic molecular techniques can establish an integrated system to clone small RNA and mRNA.RNA was extracted using TRI reagents (Sigma T9424) with a tissue tearor according to the manufacturer's protocol. Worms were grown on OP50 at 20°C for ∼70 h. To prepare single worm RNA, individual worms were transferred to a PCR tube and incubated with 0.4 µg/µL proteinase K in 10 µL buffer containing 40 mM Tris (pH 7.5), 10 mM EDTA, 0.3 M NaCl, 0.5% NP-40 and 0.5% SDS at 65°C for 10 min. The resulting RNA was phenol/chloroform extracted and coprecipitated with 20 µg glycogen.C. elegans PIR-1 gene, T23G7.5a.1, was integrated into pET-28a between the NdeI and BamHI restriction sites. The resulting plasmid was transformed into the BL21 (DE3)-RIL strain for protein expression. To express the recombinant PIR-1, a single colony was grown in 5 ml Terrific Broth (TB) medium containing 50 µg/mL Kanamycin and 20 µg/mL chloramphenicol at 37°C for 8 h; the whole culture was inoculated into 1 L TB media containing 50 µg/mL Kanamycin and 20 µg/mL chloramphenicol to grow ∼24 h at room temperature, reaching OD600 0.5; and the protein expression was induced using 0.5 mM IPTG at 16°C for 15 h. The cells were pelleted and resuspended in 25 mL lysis/wash buffer containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM 2-mercaptoethanol, 5% glycerol, 10 mM imidazole, 0.01% NP40, and 1 mM PMSF. The cells were sonicated on ice using 30 cycles of 20-sec sonication followed by 40-sec pause. The resulting solution was centrifuged at 20,000g at 4°C for 10 min and the supernatant was mixed with 2.5 mL HisPur Ni-NTA (Thermo Fisher Scientific) beads, which were prewashed three times with the lysis/wash buffer without PMSF. After 1-h nutation at 4°C, the beads were washed with 200 mL lysis/wash buffer in a 20 mL column and the protein was eluted using 10 of 0.5 mL lysis/wash buffer containing 0.4 M imidazole. The fractions 5–7, which contained the majority of the protein, were pooled and loaded onto a HiPrep Sephacryl S-100 HR column and fractionated with the imidazole-free lysis/wash buffer using the NGC Quest 100 Plus Chromatography System (Bio-Rad). The FPLC fractions contained a recombinant PIR-1 of high homogeneity . The pooled fractions were dialyzed for storage at −80°C using a buffer containing 20 mM Tris (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.01% NP-40, 0.1 mM EDTA, and 50% glycerol.The coding sequence of T4 DNA ligase was used to adenylylate a 3′ linker oligo 5′p-AGATCGGAAGAGCACACGTCTGAACTCCAGTCA/ddC/, which together with 5′ACGGCATACGAGGGAAG/ddC/ was annealed to 5′CTCTTCCGATCTGCTTCCCTCGT A/ddC/ at 95°C for 2 min followed by a slow cooling (0.1°C/sec) to room temperature, forming a nicked double-stranded DNA at 10 µM concentration in 10 mM Tris pH 7.5 and 30 mM KCl. Then 2.5 µM of the annealed DNA was incubated with 2.5 µM of T4 DNA ligase in 1× DNA ligation buffer at 37°C for 1 h, followed by phenol extraction, DNA precipitation and purification using a 15% PAGE/7M urea gel.2 at 60°C for 10 min, generating RNA fragments with a median size of ∼150 nt.A total of 8–2000 ng total RNA was partially digested with 0.01 unit of nuclease P1 in a buffer containing 50 mM sodium citrate (pH 7.0) and 10 mM MgClC. elegans analyses, reads were mapped to the genome (WormBase release WS215) and the Generic Genome Browser was used to visualize the alignments  scripts and Bowtie 0.12.7 . For C. ignments . The sofSupplemental material is available for this article.


Oryza sativa L.) growth, accumulation and distribution of Mn, accumulation of iron (Fe), zinc (Zn) and copper (Cu), root cell wall components, and expression of Mn and Fe transporter genes. After rice seedlings were grown in non-pH-buffered nutrient solution for 2 days, the pH of growth medium changed from an initial value of 4.5 to 3.5 and to 5.5 in the presence of OsNramp5 in roots under non-pH-buffered conditions, but not under pH-buffered ones. OsNramp5 expression was down-regulated at pH 3.5 compared with pH 5.5. Another efflux Mn transporter gene, OsMTP9, was not regulated by either N form or pH. High pH (5.5) enhanced the expression of the Fe transporter gene OsIRT1 and increased the accumulation of Zn but not Fe or Cu in shoots compared with pH 3.5. Taken together, our results indicate that OsNramp5. In addition, the up-regulation of OsIRT1 expression may contribute to the increased Zn uptake by rice at high pH of nutrient solution.Ammonium ( As a cofactor, Mn plays an important role in activating approximately 35 different enzymes . Excess o plants . Dependis  (Holcus lanatus and Bromus erectus (Cucumis melo L.) (Tagetes erects L. and T. patula L.) (Picea abies L. Karst.) (2+ uptake (Nitrogen (N) is an essential macroelement required by plants and is taken up by roots in two main forms, ammonium (e (NO3−) . In acidtilizers . Previouvulgare) , Holcus  erectus , muskmelmelo L.) , marigoltula L.) , and Nor Karst.) . Althoug+ uptake , the exaOryza sativa L.) is an important staple food crop consumed by nearly half of the world’s population  in a black plastic container. After 7 days, uniform seedlings were transferred to a 3.2-L black pot containing nutrient solution and cultivated in a controlled environmental growth chamber under a 14-h/10-h (30°C/25°C) day–night cycle and a relative humidity of 65% for 7 or 19 days. The nutrient solution was a modified full-strength Kimura B solution (pH 4.5) containing 0.5 mM NH4NO3, 0.18 mM KH2PO4, 0.55 mM KCl, 0.54 mM MgSO4·7H2O, 0.36 mM CaCl2·2H2O, 0.5 µM MnCl2·4H2O, 3 µM H3BO3, 1 µM NaMoO4·2H2O, 0.4 µM ZnSO4·7H2O, 0.2 µM CuSO4·5H2O, and 20 µM Fe(III)-EDTA. The solution was renewed every 2 days. Rice seedlings (14 or 26 days old) were treated with 1 mM 4Cl and NaNO3, respectively. Manganese was supplied as MnCl2·4H2O. All experiments were conducted with three to four biological replicates.We used 2+ in different N forms for 1 and 3 days, the roots were sampled for the expression analysis of Mn transporter genes. The solution was renewed after 1.5 days. After treated with 0.5 or 500 µM Mn2+ in different N forms for 10 days, the roots were used for the extraction and measurement of cell wall polysaccharide fractions. After treated with 0.5, 200, 500, or 1,000 µM Mn2+ in different N forms for 14 days, the roots were washed three times with cold 5 mM CaCl2 solution and separated from the shoots for dry weight measurements and Mn determination. The solution was renewed every 2 days.Rice seedlings (14 days old) were grown in a modified full-strength Kimura B nutrient solution (pH 4.5) containing 1 mM 2+ with 1 mM 2 solution and separated from the shoots for Mn, Fe, Zn, and Cu determination. The roots were also sampled for the expression analysis of Mn and Fe transporter genes.Rice seedlings (14 days old) were cultured in a modified full-strength Kimura B nutrient solution containing 0.5 or 500 µM Mn2+ with 1 mM Rice seedlings (26 days old) were cultured in a modified full-strength Kimura B nutrient solution containing 500 µM MnIn the above experiments, the nutrient solution was adjusted to a pH of 3.5 or 5.5 every 12 h by addition of 0.1 M HCl or NaOH and renewed at 36 h.g for 10 min. The residual cell walls were washed three times with 70% ethanol every 5 min and then dried at 70°C for 3 days. Mn concentrations in root cell sap, cell walls, and xylem sap were determined as described below.The shoots (2 cm above the root) were decapitated with a razor, and xylem sap was collected with a micropipette for 1 h. The excised roots were placed in a filter with 0.45 µm microporous membrane in a tube  and frozen at −80°C overnight. After thawing to room temperature, root cell sap was obtained by centrifugation at 20,600 ×3 at 140°C. After appropriate dilution, the concentration of Mn and Fe in roots and shoots was determined by Inductive Coupled Plasma Optical Emission Spectroscopy (ICP-OES) , and that of Zn and Cu was determined by Inductive Coupled Plasma Mass Spectroscopy (ICP-MS) . The Mn concentration in root cell sap, xylem sap, and digested cell walls was also determined by ICP-MS.Dried samples were digested with concentrated HNOTaq (Takara). Primer sequences used for amplification of OsNramp5 were 5’-CAGCAGCAGTAAGAGCAAGATG-3’ (forward) and 5’-GTGCTCAGGAAGTACATGTTGAT-3’ (reverse); 5’-AGGACCATTTCTTCGACGTG-3’ (forward) and 5’-TCCATCCACCATTTGTACCG-3’ (reverse) for OsMTP9; 5’-CGTCTTCTTCTTCTCCACCACGAC-3’ (forward) and 5’-GCAGCTGATGATCGAGTCTGACC-3’ (reverse) for OsIRT1. HistoneH3 was used as an internal standard with primers pairs 5’-AGTTTGGTCGCTCTCGATTTCG-3’ (forward) and 5’-TCAACAAGTTGACCACGTCACG-3’ (reverse). The relative expression was normalized by the ΔΔ Ct method.Total RNA from rice roots was extracted using an RNeasy Plant Mini kit (Qiagen) and converted to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo) following the manufacturer’s protocols. The quantitative PCR was performed on a LightCycler 480 Instrument  using SYBR premix Ex 4 at room temperature, and the supernatants were used as the hemicellulose fraction. In accordance with previous studies, the uronic acid content of the pectin and hemicellulose fractions was determined spectrophotometrically at 520 and 490 nm, respectively  was analyzed by Duncan’s test.All experiments were repeated independently at least two times with three to four biological replicates, and a representative set of data is presented in the At a Mn concentration of 0.5 µM, shoot dry weights were similar under Root and shoot Mn concentrations and Mn uptake were remarkably higher under In the above experiment, the nutrient solution pH decreased from an initial value of 4.5 to 3.5 in the presence of To examine whether pH affects Mn distribution to different organs, we determined Mn concentrations of different organs and calculated distribution ratios at pH levels of 3.5 and 5.5 in the presence of Mn concentrations in root cell sap and xylem sap were much higher at pH 5.5 than at pH 3.5 regardless of N form, but were similar between OsNramp5 and OsMTP9, in roots exposed to non-pH-buffered or buffered solutions with different N forms and Mn concentrations. After 1-day exposure, the expression level of OsNramp5 was similar between OsNramp5 was up-regulated by OsMTP9 between OsNramp5 was up-regulated at pH 5.5 relative to pH 3.5 buffered with 5 mM Homo-PIPES regardless of N form and Mn concentration after 3-day exposure, but was unaffected by the form of N at a given pH  in rice . In agreOsNramp5. In addition, high pH up-regulated the expression of OsIRT1 and increased Zn uptake by rice.Our results indicate that All datasets [GENERATED/ANALYZED] for this study are included in the manuscript and the supplementary files.AH and XZ conceived and designed the experiments. AH performed most of the experiments. MZ and LS conducted the collection and determination of root cell sap and xylem sap. AH, XZ, and RS analyzed data, prepared the figures, and wrote the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.This work was supported financially by the National Natural Science Foundation of China (No. 31672229), the Key Research Program of the Chinese Academy of Sciences (KFZD-SW-112-01-09), the Research Startup Foundation of Nantong University (No. 03081206), and the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB15030202).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.


The inhibitory effect of dAbs on TNF-α biological effect was determined in MTT assay in which I44 and I49 prevented TNF-α cell cytotoxicity with IC50 values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-α dAbs could bind to and inhibit TNF-α activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-α. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-α antibodies.Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. The current study aimed to investigate the binding abilities of I44 and I49 domain antibodies to TNF-α. The dAbs were expressed in bacterial expression system and purified by affinity chromatography using Ni-sepharose column. The expression and purity of the proteins were evaluated using western blotting and SDS-PAGE techniques, respectively. ELISA experiment showed that I44 and I49 dAbs bind to TNF-α with the binding constants (K To gain insight into the mode of interactions of the identified anti-TNF-α dAbs with TNF-α, molecular docking study was performed. Cytokines, as a result of inflammation, lie at the heart of chronic inflammatory and autoimmune diseases. Tumor necrosis factor alpha (TNF-α) is one of the crucial cytokines, as it directly initiates the inflammatory processes and also activates the other inflammatory cytokines . Althougic cells . TNF-α iic cells , 4. At tic cells . Rheumatic cells . Beside ic cells , 11. Theic cells -16. Althic cells , 17-19. ic cells . Antibodic cells , 22. In ic cells . The purReagents Tryptone, yeast extract, isopropyl-D-thio galactopyranoside (IPTG), Triton X-100, potassium acetate, phenylmethylsulfonyl fluoride (PMSF), and N,N,N′,N′-tetra methyl ethylene diamine (TEMED), were purchased from AppliChem . 3,3′,5,5′-Tetramethylbenzidine (TMB),Acrylamide, N,N′-methylene-bis-acrylamide, and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich, USA. Mouse anti-His primary antibody was prepared from GE Healthcare (Sweden). Goat anti-mouse IgG-HRP secondary antibody was purchased from Santa Cruz Biotechnology (USA). BM Chemiluminescence Western Blotting kit was purchased from Roche Diagnostics GmbH . Adalimumab was a kind gift from CinnaGen Company, Iran. All chemicals and reagents were of molecular biology grade. Ultra pure water, produced by Milli-Q system , was used for preparation of all solutions. Expression and purification of anti –TNF-α domain antibodiesTomlinson I and J scfv libraries were used for identification of specific anti-TNF antibodies as described in our previous study in which phage harboring I44 and I49 dAbs demonstrated high affinity towards TNF-α . E. coli origami(DE3) was infected by phage particles harboring I44 and I49 coding sequences. To do this, from each serially diluted phage, 10 µL was added to 100 µL E. coli origami(DE3) at OD 0.4 followed by incubation at 37 °C for 30 min without shaking. Then the mixtures were poured onto LB plates supplemented with 100 µg/mL ampicillin and 1% glucose. In the next day, a single colony of E. coli origami (DE3), infected with phage, was inoculated into 10 mL LB-ampicillin medium and cultured overnight at 37 °C. The overnight culture was diluted 1:50 in 500 mL LB medium and incubated at 37 °C while shaking. At OD of 0.9, IPTG with final concentration of 1 mM, was added and the temperature was set to 30 °C for overnight incubation. Then the culture was centrifuged at 3,000×g for 15 min and the harvested bacterial pellet was resuspended in lysis buffer . The suspension was freeze-thawed three times using liquid nitrogen followed by sonication five times on ice at 60% pulse for 30 sec with 30 sec intervals. Bacterial debris was removed by centrifugation at 8,0002PO4 pH 7.4, and NaCl 500 mM. Finally, the purified proteins were dialyzed against Tris 50 mM pH 8, NaCl 150 mM buffer, and analyzed on SDS-PAGE to monitor the protein production and purification. Western blotting technique was used to verify the expression of antibodies in which mouse anti-His primary antibody (1:3000 diluted in BSA) and goat anti-mouse IgG-HRP secondary antibody (1:8000 diluted in BSA) were used for protein detection. The protein bands were visualized by applying enhanced chemiluminescence (ECL) reagent.In order to express I44 and I49 proteins, ELISA experiment2SO4. The absorbance was measured at 450 nm and 650 nm using ELISA plate reader. TNF-α uncoated wells and TNF-α coated wells without addition of dAb were used as the controls. All data points were the average of three individual experiments. The results were fit into one-site specific binding curve using Prism program  in order to calculate the Kd values.The functionality of the produced dAbs was tested by ELISA experiment. To this end, TNF-α (100 µg/mL) prepared in house as described previously in , was coaMTT assay2. Different concentrations of the affinity purified dAbs (ranging from 0.023 to 1.83 µM for I44 and 0.047 to 3.82 µM for I49) were added to 0.0057 nM of TNF-α and the mixtures were incubated for 24 h in the presence of Actionomycin (1 µg/mL) at 37 °C. The mixtures were then added to the seeded cells and incubated for additional 24 h. Adalimumab, a marketed anti-TNF-α antibody at different concentration between 6.76E-5 and 6.76 nM, were used as the positive control. After incubation, 20 µL of MTT (5 mg/mL) was added to each well and the plate was kept in the incubator for 4 h. After discarding the supernatant, 100 µL of solublizing buffer containing Sorensen buffer (12.5%) and DMSO (87.5%) was added to each well and incubated for 40 min at room temperature with agitation. Finally, the absorbance was measured at 570 nm. For each concentration the experiment was carried out in triplicate wells. The achieved results represented as the percentages of TNF-α neutralizing effects in comparison to the TNF-α treated (maximum toxicity) and untreated cells (maximum viability) were fit into dose-response inhibition curve using Prism program .Murine L929 fibroblast cells were cultured in RPMI supplemented with 10% FBS. The cells were harvested in log phase growth using trypsin-EDTA. L929 cell suspension including approximately 10,000 cells were seeded in each well of 96-well plate and incubated for 24 h at 37 °C under a humidified atmosphere and 5% COMolecular modeling and docking studyThe crystal structure of TNF-α trimer was retrieved from protein data bank (PDB ID: 1TNF). In order to construct the 3D-structure of I44 and I49 dAbs from the corresponding amino acid sequences , the homExpression and purification of anti –TNF-α domain antibodiesE. coli origami(DE3) expression system induced by 1mM IPTG under temperature of 30 °C for overnight. The produced I44 and I49 antibodies were purified on affinity column resulting in 0.28 and 0.6 mg purified proteins, respectively  in different concentrations ranging from 0.35 to 5.83 µM were added to 96-well plate coated with TNF-α. Then, anti-6×His and HRP-conjugated goat anti-mouse antibodies were sequentially used to evaluate the TNF-α binding ability of dAbs. Analyzing of the results showed that I44 and I49 dAbs bind to TNF-α with Kd values of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively  were identified (E.coli origami (DE3) bacteria. Presence of PelB signal peptide in phage genome enables periplasmic expression of the target proteins . This feature provides an advantage for the current expression system where periplasmic compartment supplies a reduced environment suitable for the formation of disulfide bonds in 3D structures of I44 and I49 proteins . Such big difference in IC50 values can be attributed to the lower binding valence of I44 and I49 single dAbs (~17 kDa) compared to adalimumab (148 kDa). To identify the most important residues involved in antibodies-TNF-α interactions, molecular docking was carried out in which the modeled structures of dAbs were docked onto TNF-α trimer. The docking scores obtained from ZDock program were 952.23 and 981.76 for I44-TNF-α and I49-TNF-α complexes, respectively, which is in close agreement with the data achieved from ELISA experiment where I49 showed higher affinity towards TNF-α compared to I44. In the next step, the possible interactions between dAbs and TNF-α were predicted using PIC web server  and I49 Tyr53 (CDR2) with Val91 and Gly148 of TNF-α, respectively (Progress of inflammatory conditions greatly depends on TNF-α aberrant production. Therefore, prevention of TNF-α activity would result in promising outcomes in patients suffering from inflammatory diseases. In this regard much attention has been devoted to develop anti-TNF-α agents which could directly bind to TNF-α suppressing its adverse effects. These inhibitory molecules vary from small molecules such as peptides to macromolecules such as full length antibodies -42. Up tentified . Compareentified . On the entified . Based oproteins , 55. Thiproteins . The varproteins . In ELISas scFvs , 58. Theb server . Accordiof TNF-α . In addiof TNF-α . These rucidated . Apart fectively . Such inectively . Compariectively . Compariectively , 61. In the current study, the previously identified anti-TNF-α dAbs, I44 and I49, were recombinantly produced and purified. The binding properties of the prepared antibodies were determined using experimental and molecular modeling techniques. Based on experimental assays, these dAbs are capable of binding to and inhibiting TNF-α cytotoxic effects. Molecular docking studies identified the key residues important in the interactions between the studied domain antibodies and TNF-α. The information obtained in the current study can be useful for further structural studies and designing potent anti-TNF-α small format antibodies.


Ilex asprella in our previous work belong to multifunctional oxidosqualene cyclases and can produce α-amyrin and β-amyrin at different ratios. More than 80% of total production of IaAS1 is α-amyrin; while IaAS2 mainly produces β-amyrin with a yield of 95%. Here, we present a molecular modeling approach to explore the underlying mechanism for selective synthesis. The structures of IaAS1 and IaAS2 were constructed by homology modeling, and were evaluated by Ramachandran Plot and Verify 3D program. The enzyme-product conformations generated by molecular docking indicated that ASP484 residue plays an important role in the catalytic process; and TRP611 residue of IaAS2 had interaction with β-amyrin through π–σ interaction. MM/GBSA binding free energy calculations and free energy decomposition after 50 ns molecular dynamics simulations were performed. The binding affinity between the main product and corresponding enzyme was higher than that of the by-product. Conserved amino acid residues such as TRP257; TYR259; PHE47; TRP534; TRP612; and TYR728 for IaAS1  had strong interactions with both products. GLN450 and LYS372 had negative contribution to binding affinity between α-amyrin or β-amyrin and IaAS1. LYS372 and ARG261 had strong repulsive effects for the binding of α-amyrin with IaAS2. The importance of Lys372 and TRP612 of IaAS1, and Lys372 and TRP611 of IaAS2, for synthesizing amyrins were confirmed by site-directed mutagenesis. The different patterns of residue–product interactions is the cause for the difference in the yields of two products.Amyrins are the immediate precursors of many pharmaceutically important pentacyclic triterpenoids. Although various amyrin synthases have been identified, little is known about the relationship between protein structures and the constituent and content of the products. IaAS1 and IaAS2 identified from  Oxidosqualene cyclases (OSCs) belong to a multi-gene family of enzymes that convert a linear molecule of 2,3-oxidosqualene (OS) to polycyclic products, such as phytosterols or triterpenoids ,2,3. TriIlex asprella in our previous work can simultaneously synthesize α-amyrin and β-amyrin, which can be further transferred into ursane and oleanane-type triterpenoids, respectively [Saccharomyces cerevisiae, which were named IaAS1 and IaAS2. The products of IaAS1 and IaAS2 were identified and quantified by GC-MS. Both products were synthesized by IaAS1 and IaAS2. α-Amyrin was the main product of IaAS1, and the ratio of α-amyrin to β-amyrin was about 4:1. β-Amyrin was the main product of IaAS2, and the ratio of α-amyrin to β-amyrin was about 1:19 [Avena strigose [Arabidopsis thaliana [Arabidopsis thaliana [Arabidopsis thaliana [Arabidopsis thaliana [Arabidopsis thaliana [Cephalosporium caerulens [Euphorbia tirucalli [Olea europaea [Panax ginseng [Saccharomyces cerevisiae [More than 30 OSCs have been cloned and sequenced, including the synthetases of lanosterol, cycloartenol, β-amyrin, and lupinol ,14. Multectively . The twoout 1:19 . Severalstrigose , AtCYC: thaliana , AtCPI: thaliana , AtLSSl:thaliana , AtLUP1:thaliana , CcLSS: aerulens , EtAS: βirucalli ,25,26,27europaea , PgAS: β ginseng , and ScLrevisiae ,35,36,37Alicyclobacillus acidocaldarius [The sequence alignment of IaAS1 and IaAS2 revealed several amino acid (AA) residues around the active sites, which might be responsible for product specificity. The underlying mechanism of selective synthesis has not been fully elucidated. A significant cause is the lacking of known structures of OSCs. At present, only the structures of squalene-hopene cyclase from aldarius ,39,40 analdarius  have beeThe common template for IaAS1 and IaAS2 obtained from the online BLAST tool was human lanosterol synthase (PDB: 1W6K ). The teEuphorbia tirucalli L. (ETAS) [Multi-sequence alignment of the template, β-amyrin synthase of . (ETAS) , IaAS1 a. (ETAS) . It has . (ETAS) . Both CYTotal Energy is the sum of the scoring function value of all homology-derived pseudo-energy terms and stereochemical pseudo-energy terms. The DOPE Score is an atomic distance-dependent statistical potential from a sample of native structures [Total Energy indicates that the model satisfies the homology restraints better. A lower DOPE Score also indicates a better model. The PDF Total Energy and DOPE Score for the best model of IaAS1 were 4255.02 and −92,571.68, respectively. The PDF Total Energy and DOPE Score for the best model of IaAS2 were 5400.99 and −93,962.45, respectively. The structures of IaAS1 and IaAS2 were further verified using Ramachandran plots and the online server of SAVES v5.0. The Ramachandran plots indicated that most regions for backbone dihedral angles ψ against φ of amino acid residues in both structures of IaAS1 and IaAS2 were energetically allowed (The MODELER program in Discovery Studio v2.5 (DS) was used to construct the structures of IaAS1 and IaAS2 A–C. Probructures . Smaller allowed D–F. The The Verify 3D program  determinMolecular docking is a widely used structure-based drug design technique. It can predict the conformation of a ligand in the target binding site and calculate the binding energy . Both α-The four docked conformations were used as initial conformations of MD simulations. Each system was performed for 50 ns MD simulations. The structural stability of enzyme–product complex was evaluated by calculation of the root mean square deviation (RMSD). RMSD measures the deviation of a set of coordinates of a protein to a reference set of coordinates. The results showed that the complex of IaAS1 and α-amyrin or β-amyrin remained stable throughout the simulation. The complex of IaAS1 and α-amyrin or β-amyrin reached equilibrium after 35 ns . The aveThe binding free energies for the four systems were calculated based on the MD simulations by MM/GBSA method. According to the RMSD curves, five stages of MD trajectories were selected to calculate the binding free energy . A total of 5000 snapshots for each section were used for calculation. MM/GBSA results showed that the average binding free energy of IaAS1 with α-amyrin and β-amyrin was −29.38 ± 1.02 kcal/mol and −25.10 ± 0.67 kcal/mol. The average binding free energy of IaAS2 with α-amyrin and β-amyrin was −21.95 ± 0.84 kcal/mol and −31.54 ± 1.18 kcal/mol . The ordIn order to further elucidate key AA residues which had more influence on binding free energy, per-residue decompositions were performed to generate the residue–product interaction spectra. According to the RMSD results, we selected three sections of MD trajectories  to decompose the binding free energy . Based oConserved AA residues such as TRP257, TYR259, PHE473, TRP534, TRP612, and TYR728 of IaAS1  had strong interactions with the corresponding product in all four complexes. Therefore, these AA residues would play important roles in the catalytic process.The important AA residues for each system were ranked by the average contributions to the binding free energy for three sections of MD trajectories. For the IaAS1_alpha complex, TRP612 , TYR728 (−2.05 ± 0.03), PHE473 (−2.01 ± 0.03), ILE367 (−1.58 ± 0.01), TRP417 (−1.55 ± 0.08), TYR259 (−1.55 ± 0.03), VAL482 (−1.28 ± 0.09), MET729 (−1.04 ± 0.03), TRP534 (−0.96 ± 0.05), and TRP257 (−0.92 ± 0.02) were the top ten AA residues which can form strong interaction with α-amyrin A. The toIn the IaAS2_alpha complex, TYR259 (−2.05 ± 0.44), PHE727 (−1.86 ± 0.23), TRP533 (−1.40 ± 0.20), PHE473 (−1.33 ± 0.13), TRP257 (−1.17 ± 0.11), TYR560 (−1.12 ± 0.10), PHE551 (−0.97 ± 0.06), LEU733 (−0.90 ± 0.28), PHE125 (−0.89 ± 0.25), and GLU371 (−0.86 ± 0.23) contributed more to the binding free energy C. The keTrp612 was the most important AA residue for the binding of α/β-amyrin to IaAS1, while Lys372 was not conducive to the binding of β-amyrin to this enzyme. Regarding IaAS1, similar situations occurred for Trp611 and Lys372 . MoreoveMutating the Lys372 to glycine and the Trp612 to phenylalanine abolished the catalytic ability of IaAS1; none of α-amyrin or β-amyrin was detected in the metabolite. It demonstrated that Lys372 and Trp612 were essential for the biosynthesis of amyrins. While for IaAS2, Lys372G and TRP611F mutants had a different impact on the biosynthesis of α/β-amyrin . Both muhttps://www.ncbi.nlm.nih.gov) was used to search template structure (PDB ID:1w6k [http://servicesn.mbi.ucla.edu/SAVES).The sequences of the two amyrin synthases (IaAS1 and IaAS2) were retrieved from the PubMed. There were 762 AA residues in IaAS1 (GenBank: AIS39793.1) and 761 AA residues in IaAS2 (GenBank: AIS39794.1). The NCBI online Standard Protein BLAST tool ( ID:1w6k ) for hom6, 2.7× 105, and 0.02, respectively. Other parameters were set to default.The AutoDock v4.2.6 and AutoDocktools v1.5.6 (ADT)  were use+ ions were used to neutralize the solution. The fully solvated system was then minimized in 3 stages by the sander program. The first stage involved the minimization of 2000 steps of steepest descent followed by 2000 steps of conjugate gradient with the constraining of all atoms of protein and ligand. In the second stage, only the atoms of backbone were constrained to minimize water and the side chains of protein by using the steepest descent minimization of 5000 steps followed by a conjugate gradient minimization of 5000 steps. In the third stage, the entire system was optimized without any constraint and the method was the same as with the second stage. The system was then heated gradually from 0 to 310 K in the NVT ensemble and equilibrated at 310 K for another 20 ps. Then 50 ns MD simulations were performed by the pmemd program. The coordinates were saved every 10 ps for subsequent analysis. The cpptraj program was used to calculate the root mean square deviation (RMSD).The docking conformation with lowest binding energy was selected as the initial conformation for MD simulation. Gaussian 09 and antechamber programs were used to create the force field parameters for α-amyrin and β-amyrin. The ff14sb protein field  was usedbind) for the four systems was calculated using MM/GBSA methods by the MMPBSA.py program [It was reported that the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) showed better performance in ranking the binding affinities for systems without metals than the Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) ,51,52. T program  in AMBER program ,55,56. TIn order to verify the credibility of the molecular docking and molecular dynamics modeling results, site-directed mutagenesis was carried out for IaAS1 and IaAS2, respectively. Two single mutants for IaAS1 (K372G or W612F) and IaAS2 (K372G or W611F) were obtained via inverse PCR (iPCR) . BrieflyIlex asprella. On the basis of molecular docking conformations and visualized enzyme-product interactions, ASP484 was found to play an important role in the catalytic process, and TRP611 of IaAS2 had interaction with β-amyrin through π–σ interaction. The binding free energies predicted by molecular docking were consistent with the experimental results. Molecular dynamics simulations and MM/GBSA binding free energy calculations further strengthened the evidence. The results of binding free energy decomposition demonstrated that conserved AA residues such as TRP257, TYR259, PHE473, TRP534, TRP612, and TYR728 for IaAS1  had strong interactions with the corresponding product in all four complexes. Residues would have negative contributions to binding affinity between the enzymes and products. Eleven AA residues of IaAS1 and thirteen AA residues of IaAS2 which have been identified as important AA residues are consistent with the results of site-directed mutations. These two enzymes also have uniquely important residues, such as ILE367, TRP417, TYR728 for IaAS1 and GLU358, GLU371, PHE727 for IaAS2. The different patterns of residue–product interactions play an important role in the enzymatic cyclization mechanism and are responsible for product determination. The importance of Lys372 and TRP612 of IaAS1, Lys372 and TRP611 of IaAS2 for synthesizing amyrins was confirmed by site-directed mutagenesis. The results give an insight into the mechanism that OSCs convert a single substrate into different products.Both α-amyrin and β-amyrin can be synthesized by two multifunctional OSCs, but the yields for the two products differ greatly. The ratios of α-amyrin to β-amyrin produced by IaAS1 and IaAS2 are 4:1 and 1:19, respectively. In this work, a molecular modeling approach by combining homology modeling, structure evaluation, molecular docking, molecular dynamics simulations, MM/GBSA binding free energy calculations, and free energy decomposition was adopted to explore the mechanism of selective synthesis of amyrins by two oxidosqualene cyclases of