Effective transfer of genetic information during cell division requires a major reorganization of chromosome structure. This process is triggered by condensin, a conserved pentameric ATPase essential for chromosome condensation. How condensin harnesses the energy of ATP hydrolysis to promote chromatin reorganization is unknown. To address this issue, we performed a genetic screen specifically focused on the ATPase domain of Smc4, a core subunit of condensin. Our screen identified mutational hotspots that impair condensin’s ability to condense chromosomes to various degrees. These mutations have distinct effects on viability, genome stability, and chromosome morphology, revealing unique thresholds for condensin enzymatic activity in the execution of its cellular functions. Biochemical analyses indicate that inactivation of Smc4 ATPase activity can result in cell lethality because it favors a specific configuration of condensin that locks ATP in the enzyme. Together, our results provide critical insights into the mechanism used by condensin to harness the energy of ATP hydrolysis for the compaction of chromatin. In eukaryotes, the deletion of a single copy of most genes shows little or no detectable phenotype under standard proliferative conditions. This implies that a large reduction in the level of a gene product can be tolerated by eukaryotic organisms and that a “reserve capacity” is built in the protein machinery that drives most cellular processes. Here, we test if the main effector of chromosome condensation—the condensin complex—operates with a reserve enzymatic capacity in the execution of its multiple functions in vivo. To achieve this, we created an allelic series of mutations that selectively inactivate condensin ATPase activity in a graded manner. We show that many core functions of condensin can be maintained even at low levels of ATPase activity. Our data also reveal the existence of various thresholds of ATPase activity that are necessary and sufficient for the execution of different cellular functions by condensin. Notably, loss of genome stability at repetitive DNA is only observed when condensin ATPase activity is severely impaired. Taken together, our results reveal key insights into the process of ATP hydrolysis by condensin and how the energy it releases promotes genome remodeling and stability during cell division. Chromosomes must undergo a major structural reorganization to allow the efficient and error-free segregation of genetic information during mitosis. This process is known as chromosome condensation and is promoted by the condensin complex –3. The cThe catalytic core of the condensin complex is formed by two SMC family subunits, a group of proteins involved in sister chromatid cohesion, DNA repair, and chromosome condensation . SMC The functional relevance of ATPase activity has also been studied in other SMC complexes, most notably in cohesin. This complex, composed of a catalytic core of Smc1 and Smc3 proteins, is required to hold the sister chromatids together during mitosis and meiosis . In coheWith respect to condensin, it is known that ATP hydrolysis plays an essential role in its function, as the abrogation of this activity leads to cell inviability ,19. Moresmc4 mutants specifically defective in condensin ATPase activity.Ultimately, a key question for chromosome biology is how condensin uses the energy of ATP hydrolysis to convert amorphous chromatin into highly compacted chromosome in vivo. Since all ATPase-specific mutants tested so far in condensin are inviable , it has been difficult to delineate the specific contribution of condensin ATP hydrolysis to the process of chromosome condensation. To address this important question, it is necessary to identify weakened or hypomorphic alleles of condensin that, despite being catalytically active, are defective enough to stabilize intermediate states in the process of chromosome compaction. To achieve this goal, we report here the characterization of an extensive collection of The ABC-type ATPase domain is a characteristic feature of SMC proteins . This faThe role of most ABC-specific ATPase motifs has not been extensively characterized in SMC proteins. In contrast, the specific contribution of these motifs to the mode of action of ABC transporters is well documented (reviewed in ). These S. cerevisiae Smc1 (ScSmc1) , and a high-resolution structure is available for its ATPase head domain. Overlap of known SMC structures from ScSmc1 and P. furiosus Smc (PfSmc) illustrates a high degree of conservation in structural organization within this family of proteins, even when primary sequence identity is weaker than that shared by Smc1 and Smc4   30,35].,35.S. ceSMC4, we used a gene replacement strategy in S. cerevisiae. Specifically, we constructed a series of heterozygous ATPase mutants in diploid cells and uncovered the phenotype associated with these smc4 mutations after sporulation . Several other mutants showed defects only at high temperature (37 °C), including strong , intermediate (smc4-L1335A and smc4-Q1377A), and mild defects  to condense the ribosomal DNA (rDNA) locus in a nocodazole-induced early-mitotic arrest using fluorescence in situ hybridization (FISH). The phenotype of these cells was compared to that of wild-type SMC4 and cells carrying smc4-7A and smc4-22, moderate and strong mutants of condensin, respectively A; smc4-K165M; smc4-K165A; smc4-Y167C; smc4-K191M; smc4-R210A; smc4-R210M; smc4-R210K; smc4-R210D; smc4-R210P; smc4-R210A-Q211A; smc4-R210D-Q211E; smc4-Q302E; smc4-Q302K; smc4-Q302L; smc4-Q302D; smc4-S1324R; smc4-S1324N; smc4-K1328A; smc4-L1333A; smc4-L1335A; smc4-L1333A-L1335A; smc4-L1335G; smc4-L1333V-L1335A; smc4-L1333I-L1335A; smc4-P1346G; smc4-P1346T; smc4-P1346L; smc4-P1344G-P1346G; smc4-D1351A; smc4-D1351N; smc4-E1352Q; smc4-E1352D; smc4-D1358H; smc4-D1358N; smc4-D1358E; smc4-I1364N; smc4-V1365M; smc4-V1365F; smc4-Q1377A; smc4-Q1377R; smc4-A1376S-Q1377A; smc4-Q1377A- F1378A; smc4-L1383A; smc4-L1383I; smc4-L1383V; smc4-N1386H; smc4-N1386D; smc4-G1396Q; smc4-G1396P. Mutant alleles of SMC4 were introduced at the endogenous locus by transformation of digested plasmids, thus releasing a fragment containing SMC4 ORF with its selection marker. The backbone of the plasmid was lost during the process. All the mutations were confirmed by sequencing the SMC4 locus. For the overexpression of condensin subunits , PGAL1-10 or PGAL7-driven subunits  were subcloned in tandem on 2 multicopy plasmids .Mutations were introduced in Smc4 subunit using QuikChange Multi Mutagenesis kit (Stratagene) in the plasmid YCplac111-4 buffer pH 6.4 containing 3.7% formaldehyde for 2 hours at 23 °C. The procedure to analyze the morphology of the rDNA locus is based on published data ATP . The mixtures were transferred to a parafilm wrap on ice and were exposed to UV radiation (254 nm) for 10 minutes to induce cross-link. After cross-linking, protein samples were separated by SDS-PAGE, and the radioactive signal was detected using a Typhoon FLA-9500 Bio-Image Analyzer. The ATP-bound signal detected was normalized to the protein signal detected in the Coomassie-stained gels.Binding of ATP to yeast condensin was performed using the UV cross-linking procedure as previously described , with fen is the anisotropy for each protein concentration, rmax is the anisotropy at the highest protein concentration, and r0 is the anisotropy without protein. To calculate the equilibrium association constant (Kd), the normalized fluorescence anisotropy was represented as a function of protein concentration, and a curve was fit with nonlinear regression algorithm using Prism. The DNA–protein binding constant was calculated in 3 experiments performed with the same batch of purified protein.Fluorescence anisotropy experiments were carried out at 10 nM 6-FAM ssDNA and variable concentrations of protein (0.001–6 μM). Anisotropy was measured 20 minutes after the incubation of the binding reaction at 30 °C in a microplate reader (BioTek) at room temperature (λ absorbance 485 nm and λ emission 525 nm). Normalized fluorescence anisotropy (“FA”) was calculated according ton is the anisotropy for each DNA concentration, rmax is the anisotropy at the lowest ssDNA concentration, and r0 is the anisotropy with the same ration of 6-FAM ssDNA and not labeled DNA.To analyze the DNA binding reversibility, we performed a DNA competition assay. First, 10 nM of 6-FAM DNA was incubated with 500 nM of each condensin complex for 20 minutes at 30 °C. Then, the complexes were incubated with increasing concentrations of unlabeled DNA (10 nM–3 μM ssDNA) for 20 more minutes at 30 °C. Anisotropy was measured in a microplate reader (BioTek) at room temperature (λ absorbance 485 nm and λ emission 525 nm). Normalized fluorescence anisotropy (FA) for the DNA competition assay was calculated according torDNA/RDN1 copy number in yeast strains was determined by qPCR analysis of the amplification signal obtained from genomic DNA isolated from a wild-type strain (D4107), the 25xRDN1 strain (D419), and the various condensin mutants generated in this study. The RDN1 amplification signal was standardized relative to SMC4 as a gene with a single copy number using the formulaHIS3 and SUP11, as previously described ATP-labeled condensin subunits (long and short exposure), whereas the lower panel shows a Coomassie staining of the different condensin complexes used in this experiment. Data shown in this figure are from a representative experiment. (Bottom) The histogram on the left shows the relative ATP-binding activity of Smc4 with respect to that of Smc2 and Ycs4. Note that Smc2 and Ycs4 migrate at a similar position in SDS-PAGE and cannot be discriminated on standard gel, which is why the data is expressed relative to the combined signal of these two proteins. However, only Smc2 and Smc4 have the ability to bind ATP in the condensin complex. Error bars represent SD . The histogram on the right reports the quantification of ATP bound to condensin complexes normalized for protein abundance . Error bars represent SD . See (Top) The ATP-binding ability of Smc4, Smc4-K191M, Smc4-L1335A, and Smc4-Q1377A mutants were compared using equal amounts of protein. The two upper panels correspond to autoradiograms of [P(TIF)Click here for additional data file.S4 Fign = 3). See n = 3). See (A) DNA binding of Smc4, Smc4-K191M, and the H-loop and C-helix mutants was assessed as described in (TIF)Click here for additional data file.S5 Fig(A) Modeling of likely intramolecular interactions involving Leu1333 and Leu1335 within the C-helix of Smc4 and the impact of relevant mutations at those positions. Amino acid residues at position 1333 and 1335 are depicted in blue, while residues in the vicinity of these position  are depicted in red. (B) Model showing the hydrophobicity of the residues at position 1377 and 1378 in the H-loop of Smc4. Minimum hydrophobicity is depicted in blue and maximum in red.(TIF)Click here for additional data file.S6 FigSMC4 gene in human cancers. The analysis was performed using the COSMIC database [hSMC4 gene. The table on top highlights a subset of point mutations that were identified in hSMC4, how they affect hSmc4 protein sequence, their recurrence, and the corresponding residue changes in yeast Smc4. Note that N447H and S1056L mutations are not localized in evolutionarily conserved regions of hSMC4 sequence, while R1252 affects the conserved ATPase head domain of the protein. The lower part of this panel shows the growth properties of spores carrying smc4-R1384* and smc4-K1410* alleles after dissection of heterozygous diploid strains. The smc4-R1384* truncation allele corresponds to the R1252* mutation in hSMC4, whereas the smc4-K1410* truncation corresponds to a C-terminal deletion allele introduced in G. stearothermophilus SMC [SMC4 in various types of human cancers. Data are from the COSMIC database. See SMC4 relative to SMC4 gene overexpression in human cancers. See (A) Analysis of the types of mutations found in database , and mutilus SMC . (C) Ove(TIF)Click here for additional data file.S1 DataTable reporting measured and calculated values for individual experimental data shown in Figs (XLSX)Click here for additional data file.S1 TableSMC4 as control group). Star symbols signify the following p values: * p < 0.05, ** p < 0.01, *** p < 0.001, and ****p < 0.0001. N.s. indicates no significant difference, whereas a dash (–) symbol indicates not tested or not relevant.Statistical significance was calculated using one-way ANOVA with Dunnett’s post hoc test (using wild-type (XLSX)Click here for additional data file.S2 TableThe following abbreviations and symbols signify: +++, very severe defect / ++, moderately severe defect / +, weak defect / -, no detectable phenotype (or not tested) / c.s., cold-sensitive / n.r., not relevant / Y, Yes / N, No.(XLSX)Click here for additional data file.S3 TableOnly the relevant genotype differences are shown.(XLSX)Click here for additional data file.


Streptococcus pneumoniae. Intriguingly, its structural homologue, RumA, can only catalyze the methylation of U1939, while RlmC is the dedicated enzyme for m5U747 in Escherichia coli. In this study, we describe the structure of RlmCD in complex with its cofactor and the RNA substrate containing U747 at 2.00 Å or U1939 at 3.10 Å. We demonstrate that multiple structural features collaborate to establish the dual enzymatic activities of RlmCD. Of them, the side-chain rearrangement of F145 was observed to be an unusual mechanism through which RlmCD can discriminate between U747- and U1939-containing RNA substrate by switching the intermolecular aromatic stacking between protein and RNA on/off. An in-vitro methyltransferase assay and electrophoretic mobility shift assay were performed to validate these findings. Overall, our complex structures allow for a better understanding of the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of the RumA-type enzyme and the potential development of antibiotic drugs against S. pneumoniae.Methyltransferase RlmCD was previously shown to be responsible for the introduction of C5 methylation at both U747 and U1939 of the 23S ribosomal RNA in  Streptococcus pneumoniae.Methylation of nucleotides widely occurs on ribosomal RNAs (rRNAs) in all living organisms; some clusters at the functional centers of rRNA are important for protein translation. In pathogenic bacteria, it has been shown that modulation of the methylation state of some rRNA nucleotides serves as a clinically important mechanism to confer resistance to ribosome-targeting antibiotics. Because some methyltransferases can introduce methylation at different target sites, understanding the functional roles of each individual methylation site in rRNA has become meaningful but remains challenging. In this research, using protein-RNA crystal structures, we demonstrate that multiple structural determinants collaborate to establish the dual enzymatic activities of RlmCD, which has been previously shown to catalyze C5 methylation at both U747 and U1939 of 23S rRNA in some gram-positive bacteria. This structural biology study provides a new perspective for us to understand the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of RumA-type enzyme and the potential development of antibiotic drugs against  E. coli, respectively, with a variety of methylation types including 1-methylguanosine (m1G), 3-methyluridine (m3U), 5-methyluridine (m5U), 5-methylcytidine (m5C) and 2-methyladenosine (m2A) . In. In29]. To uncover the structural features determining the multi-selectivity of RlmCD for RNA substrates, further work has been conducted for this report to obtain the crystal structures of RlmCD in complex with re-designed U747 RNA substrates and the U1939 RNA substrate. Through careful structural comparison between RlmCD complexed with U747 and U1939 RNA substrates, we identified multiple crucial residues that behave differently in two specific recognitions. Further in-vitro MTase and EMSA assays were employed to confirm our observations from the structures. The structural insights we gained in this research provide a new perspective for us to understand the delicate mechanism that RlmCD employs to recognize different RNA substrates.740GGCACGUm5UGAAAAGUGCC757-3′) analogous to the major portion of the U747-methylated 23S rRNA hairpin 35 in S. pneumoniae  or 12-nt  U747-containing RNA analogue (743C-G754 and 744C-G753) was therefore crystalized with the construct of 1–454 of RlmCD containing a E443Q mutation, using S-adenosyl methionine (SAM) as the cofactor. The 743–754 base-pair in U747SL was modified to a G-C from an A-U to increase the stability of the RNA structure. A similar RNA design was also applied to co-crystalize the RlmCD with SAM and an 8-nt U1939-containing RNA stretches    to underSL complex structure crystalized in space group P1, each asymmetry unit contains two complex molecules that pack together through RNA-RNA intermolecular base stacking  as the search model , and thestacking . All strα are 0.997 Å for U747L complex and 0.995 Å for U1939L complex), without obvious relative motion between different domains upon the RNA binding  in RlmCD-SAH-U747SL show no obvious interaction with RlmCD, extending outward from the RNA catalytic groove. Given the higher resolution of RlmCD-SAH-U747L complex structure, further analysis of protein-RNA interactions for U747-containing RNA substrates was mainly based on the RlmCD-SAH-U747L ternary complex. Interestingly, in the RlmCD-SAH-U1939L complex structure, the U1939-containing RNA substrate also adopts a very similar conformation to that of U747L, even though their sequences are totally different  from the C-terminal catalytic domain by either a hydrogen bond or face-to-edge aromatic interaction. The first three residues and E443 are all highly conserved in RumA-like enzymes . BecauseL. The influence on the MTase activity and RNA binding of RlmCD by R127 and Q162 was therefore investigated by site-directed mutagenesis  binds at the catalytic groove formed by the central and catalytic domains of RumA, while the 3′-side hairpin (G1945-C1961) binds in the cleft formed between the N-terminal OB fold domain and the catalytic domain [L structure we determined in this research contains only the A1936-U1943 of 5′-end loop, we therefore focused on a comparison of the RNA binding within the catalytic grooves of RumA and RlmCD. The majority of nucleotides within A1936-U1943 maintain a very similar conformation in the two complex structures, except A1936 and A1937  that are responsible for its dual enzymatic activity towards C5 methylation of U747 and U1939, especially when these residues are all strictly conserved in YefA .L. Considering the opposite effects of Q162A mutation on MTase activities of RlmCD for U747 and U1939 RNAs, we concluded that although Q162 may not seem to be an evolutionary mutation beneficial for RlmCD MTase activity towards U1939, it does establish the binding specificity of RlmCD for U747-containing RNA.Of these residues, N249 behaves differently in interactions of RlmCD and two different RNA substrates but with a more significant impact on U747 methylation, confirming our previous conclusion that the linker containing N249 is a newly evolved structural determinant that helps RlmCD to obtain additional MTase activity for U747 . As anotL and U1939L show that F145 adopts multiple conformations for its side-chain under different circumstances , and RumA (F148). Unlike the RumA structures in which the conformations of F148 side-chain remain almost the same with and without RNA substrates, the structures of RlmCD in the absence and presence of U747mstances . Upon biL and U1939L unveil multiple structural features to explain how RlmCD can specifically recognize U747 RNA while it remains enzymatically functional for the methylation of U1939. The aromatic side-chain rearrangement of F145 was observed to be an unusual strategy used by RlmCD to discriminate between two different sites on 23S rRNA by switching on/off the intermolecular π-π stacking between protein and RNA; dit Konté and coworkers recently reported a similar mechanism employed by RRM3 (RNA recognition motif 3) of CUG-BP2 (GUG triplet repeats RNA binding protein) to discriminate its two U-rich RNA substrates [Overall, our crystal structures of RlmCD complexed with U747bstrates . Both th5U RNA MTases evolved from a common ancestor, which is most likely an RumA-like enzyme that originated amongst bacteria [E. coli m5U MTase, RlmC and TrmA [It has been suggested that all contemporary mbacteria . RumA hoand TrmA , are resand TrmA . In receand TrmA .5U sites on 23S rRNA unique to B. subtilis and S. pneumoniae, or is this feature prevalent in gram-positive bacteria? Based on our structural findings in this research, a further bioinformatics study should be employed to provide hints to answer this question.Although both RlmCD and RlmC can introduce C5 methylation at U747 of 23S rRNA, their RNA-recognition mechanisms may differ, as the residues contributing to the enzymatic activity of RlmCD for U747  are not conserved in RlmC, and RlmC does not possess a TRAM domain . Most liS. pneumoniae is the major cause of bacterial pneumonia and meningitis, as well as bloodstream, ear, and sinus infections. Due to the global antibiotic resistance crisis, it has been estimated that ~1.2 million illnesses and ~7,000 deaths per year are caused by S. pneumoniae infection. Its drug resistance has been listed as one of serious threats to human health by the CDC [5U747 locates in the loop region of hairpin 35, protruding into the large subunit tunnel for extension of the nascent peptide. Although U747 methylation has been recently found to contribute to TEL susceptibility through promoting efficient RlmAII-mediated G748 methylation in S. pneumoniae, its precise functional role must be further illustrated. The m5U747 is close to NPET and itself may become a target for the design of new antibiotics against diseases and syndromes induced by S. pneumoniae. the CDC . In 23S 5U747 in S. pneumoniae or other related pathogens [In this research, although multiple residues were shown to contribute simultaneously to the specific recognitions of RlmCD for two different sites on 23S rRNA, the single mutant F145A alone can abolish the MTase activity of RlmCD for U747 while its enzymatic activity for U1939 remains almost intact, exhibiting a relatively ‘clean’ effect in decoupling the dual MTase activities of RlmCD; however, this result must be further confirmed by in-vivo experiments. F145A therefore provides us with an unprecedented opportunity to illustrate the functional role of U747 methylation in cells without affecting the methylation level of U1939 in 23S rRNA under normal conditions and certain stress conditions, which may eventually assist in the development of new antimicrobial drugs targeting at mathogens .S. pneumoniae genomic DNA via PCR. The full-length RlmCD (residue 1–543) and C-terminal truncated RlmCD (residue 1–454) were subcloned into a modified pET28a (Novagen) vector with an 8×His-SUMO tag and a ULP1 cleavage site (pET-SUMO vector) at the N terminal site. All single residue mutants were generated using MutanBEST kit (TaRaKa) and verified by DNA sequencing. The plasmids were transformed into E. coli BL21-Gold (DE3) cells.The RlmCD (sp_1029) ORF was amplified from 600 reached 0.8, and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. After induction, cells were grown at 16°C for an additional 24 hours before harvesting. The cell pellets were suspended with binding buffer , and lysed by sonication. After centrifugation, the supernatant was purified using Ni-NTA affinity chromatography (Qiagen). The eluted SUMO-tag protein was loaded on Superdex 200(16/60)  equilibrated with binding buffer for further purification. The elution sample was mixed with ULP1 enzyme against storage buffer  to remove the SUMO tag. After overnight cleavage, the mixture was further purified by Superdex 200(16/60) , again with storage buffer.For wild-type and mutant protein expression, cells were grown in LB medium at 37°C until the ODL RNA (5′-745GUUGAAAA752-3′), U747SL RNA (5′-743CCGUUGAAAAGG754-3′), FAM-U747SL RNA (5′-FAM-743CCGUUGAAAAGG754-3′), U1939L RNA (5′-1935AAAUUCCU1943-3′), 30-nt FAM-U1939 RNA (5′-FAM-1933GCGAAAUUCCUUGUCGGGUAAGUUCCGACC1962-3′) were purchased from TaRaKa Bio Inc. and dissolved in diethyl pyrocarbonate (DEPC)-treated water to a final concentration of 2 mM. The 30-nt U1939 RNA (5′-1933GCGAAAUUCCUUGUCGGGUAAGUUCCGACC1962-3′) for the in-vitro MTase assay was obtained using in-vitro RNA transcription by T7 RNA polymerase, as previously described [Unmodified and 5′-6-carboxyfluorescein (FAM)-labelled RNA oligomers, including U747escribed . The nuc2 and 10% DEPC at 298 K for 1 hour.RlmCD (1–454) E443Q mutant was concentrated to ~6 mg/mL in storage buffer . RlmCD concentration was determined by its ultraviolet absorption at wavelength 280 nm. The extinction coefficient of full-length RlmCD was 0.78 and RlmCD (1–454) was 0.71. The enzyme-cofactor-RNA covalent complex was prepared by incubating 0.117 mM RlmCD with 0.234 mM SAM and the 0.129 mM RNA substrate at a molecular ratio of 1:2:1.1 in storage buffer supplemented with 1 mM MgClL for data collection were finally refined under 0.1 M HEPES, pH 7.5, 36%(w/v) PEG600; Crystals of RlmCD-SAH-747SL were finally refined under 0.2 M ammonium acetate, 0.1 M sodium acetate, pH5.2 20% (w/v) PEG3350; Crystals of RlmCD-SAH-1939L were finally refined under 0.1 M MES, pH 5.5, 0.15 M ammonium sulfate, 25% PEG4000. All the pH was adjusted at 298K.The initial crystallization trial was set up using the hanging drop vapor diffusion method at 293 K. Each sample was mixed with buffer in a 1:1 ratio to equilibrate against a 100-μL reservoir solution. The crystals started to appear after 1 week at 293 K. Crystals of RlmCD-SAH-747The size suitable crystals (fitted with 0.1–0.2 mm cryoloop) were transferred directly into cryo-protectant flash-frozen into liquid nitrogen. The cryo-protectant was prepared by mixing 7 μL crystallization buffer with 3 μL glycerol. X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) Beamline 19U1 or 17U. Intensity data were indexed and scaled by HKL2000. All complex structures were solved by molecular replacement with the program MOLREP in CCP4i using apo RlmCD (PDB ID: 5XJ1) as the search model . The RNA2. The final concentrations of FAM-labelled U747SL RNA or 30-nt U1939 RNA and SAH were 0.5 and 5μM. The final concentrations of RlmCD or its mutants are 0, 0.1, 0.2, 0.4, 0.8, 1.0, 2.5 and 5.0 μM, as indicated in 2O, 1.5 mL 40% Acrylamide: Bis-acrylamide (19:1), 0.6 mL 10X TBE buffer, 30 μL 10% APS, and 3 μL TEMED. Subsequently, gels were scanned using a Typhoo FLA 7000 biomolecular imager.EMSA was performed in a 10-μL reaction mixture containing 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, and 1 mM MgClThe MTase assay was performed as previously described .S1 FigSL (A) and RlmCD-SAH-U1939L (B). Electron density maps with 2Fo-Fc calculated at 1.0σ shown for SAH, RlmCD C408, and all RNA nucleotides. (C) The proposed catalytic mechanism of m5U MTase RlmCD.Close-up of active site in RlmCD-SAH-U747(TIF)Click here for additional data file.S2 FigSL complex, each asymmetry unit contains two complex molecules (lime and pink) that pack together through RNA-RNA intermolecular base-stacking. (B) Superimposition of complex structures of RlmCD-SAH-U747L (marine) and RlmCD-SAH-U747SL (pink) with an RMSD for Cα atoms of 0.4 Å. (Inset) Close-up of overall structures of U747L and U747SL. Orientation of structures is slightly adjusted for clarity.(A) In crystal of RlmCD-SAH-U747(TIF)Click here for additional data file.S3 FigL (A) and RlmCD-SAH-U1939L (B).Schematic view of protein-RNA interactions in RlmCD-SAH-U747(TIF)Click here for additional data file.S4 FigL (except U747) and surrounding RlmCD residues. (D) Aromatic stacking in RlmCD-SAH-U747L structure. RlmCD residues in different complex structures are shown in blue and yellow, respectively. (E-G) Interaction details between individual nucleotides of U1939L (except U1939) and surrounding RlmCD residues. (H) Aromatic stacking in RlmCD-SAH-U1939L structure.(A-C) Interaction details between individual nucleotides of U747(TIF)Click here for additional data file.S5 FigConserved residues are shown in white on a red background, and similar residues are shown in red in a blue rectangle. Conserved residues participating in U747 recognition between RlmCD and YefA  are labeled with a black asterisk.(TIF)Click here for additional data file.S6 FigFree RNA and shifted protein-RNA complex are labelled.(TIF)Click here for additional data file.S7 FigFree RNA and shifted protein-RNA complexes are labelled. Two shifted bands for protein-RNA complexes are observed in all EMSA experiments of U1939 RNA and we proposed that the upper band may be induced by the nonspecific binding of 30-nt RNA for an extra RlmCD molecule under high protein concentrations.(TIF)Click here for additional data file.S8 FigInset) Close-up of overall structures of U1939L RNAs in two models. (B) Stick model of U1939L RNA in 3.24 Å RlmCD-SAH-U1939L complex structure. 2Fo-Fc electron density map is calculated at 1.0σ.(A) Superimposition of 3.10 Å (yellow) and 3.24 Å (aquamarine) RlmCD-SAH-U1939L complex structures. ((TIF)Click here for additional data file.


Here we describe methods for determining real-time intracellular Ca2+ release and contraction in vSMCs of neural crest origin differentiated from human induced pluripotent stem cells using multiple protocols, and compare these with primary human brain vascular pericytes and smooth muscle cells. Open-source software was adapted for automated high-density analysis of Ca2+-release kinetics and contraction by tracking individual cells. Simultaneous measurements on hundreds of cells revealed heterogeneity in responses to vasoconstrictors that would likely be overlooked using manual low-throughput assays or marker expression.Vascular smooth muscle cells (vSMCs) are highly heterogeneous across different vascular beds. This is partly dictated by their developmental origin but also their position in the vascular tree, reflected in their differential responses to vasoactive agonists depending on which arteriolar or venular segment they are located. Functional assays are necessary to capture this heterogeneity   •2+ release in hiPSC-derived vSMCsMicrofluidic assay for real-time Ca•2+ release and contraction analysis in hiPSC-derived vSMCsAutomated Ca•Side-by-side comparison of hiPSC-derived vSMCs with human brain pericytes and vSMCs 2+ release and contraction in vascular smooth muscle cells differentiated from human induced pluripotent stem cells. Open-source software adapted for automated high-density analysis and simultaneous measurements of hundreds of cells revealed heterogeneity in responses to vasoconstrictors that would likely be overlooked using standard low-throughput assays or marker expression.In this article, Orlova and colleagues describe methods for real-time intracellular Ca Develop2+ . vSMCs ireuptake . Culture2+ release and contraction in vSMCs. vSMCs were differentiated via neural crest intermediates from three independent healthy hiPSC lines, using various protocols based on previously published methods  by simple mechanical elimination of the cells in the center of the colonies. FACS analysis showed that NCCs (NGFR+/HNK1+/TRA-1-60−/SOX2−) maintained their phenotype up to P7  signaling are known to be important for the induction of neural crest cells (NCCs) from human pluripotent stem cells (hPSCs) . Since tup to P7 D. Nucleaup to P7 D. Succesup to P7 E. NotablTFAP2A, SOX9, SOX10, and PAX3, and upregulation of SMC contractile markers such as ACTA2, CNN1, SM22, and MYH11, in NC-SMCs compared with NCCs; however, expression of MYH11 remained relatively low (We next differentiated NCCs into vSMCs (NC-SMCs) using previously described protocols, with some modifications as shown in vely low G. Expresvely low G. Expresvely low H and S1F2+ release was determined in NC-SMCs (differentiated with protocols A to E) and HBVPs and HBVSMCs, using an experimental setup that combined microfluidics and live-cell imaging  in two-dimensional image sequences was used (2+ release was examined upon stimulation with the vasoconstrictor endothelin-I (ET-I) (0.1 μM and 1 μM) (EDNRA), while expression of other receptors and ion channels was comparable (2+ events with a single or main peak (MP) and recurrent secondary peaks (SPs), while the vast majority of NC-SMCs differentiated using protocols C and D, as well as HBVPs, had only one MP  C. Notablnd 1 μM) C and hadmparable E. NC-SMCy one MP B and 2C.2+ signals, we derived kinetic parameters of the average fluorescence intensity of ROIs normalized to baseline over time (F/F0). Time to peak, decay, and duration were measured at the half-maximum level (F/F0)max max D. Ca2+ kexamined E. Distrireuptake E.D, which is close to zero if histograms are largely overlapping and equals 1 when histograms do not overlap at all  and kinetic parameters of Ca2+ peaks . The rationale for setting different ranges for these types of measurement was based on corresponding intrapopulation D values between either technical replicates or independent biological experiments (data not shown).To compare population histograms quantitatively, we used a method reported previously that allows measurement of the difference between two populations . A schemp at all . We clas2+ events within ROI, we obtained low D values between HBVPs and NC-SMCs differentiated with protocols C and D  but moderate to high D values between HBVPs and NC-SMCs differentiated with protocols A, B, and E and HBVSMCs (D = 0.19–0.23)  and moderate to high D values compared with NC-SMCs differentiated with protocol C and D and HBVPs (D = 0.19–0.27)    (When comparing the number of Ca19–0.23) B. HBVSMC19–0.27) B. Compar68–0.83) C but low30–0.43) D and 3E.14–0.36) C.S/S, in percent)  to reflect the extent of the differences. NC-SMCs differentiated using protocol E had the lowest difference compared with HBVSMCs (D = 0.11) and HBVPs (D = 0.21) among all NC-SMCs (D = 0.28–0.37), moderate to high differences compared with HBVSMCs (D = 0.41–0.51), and low median relative cell-surface area decreases. Additionally, side-by-side assessment of contraction upon stimulation with ET-I (0.1 μM) in NC-SMCs differentiated using protocols A to E from three independent hiPSC lines showed reproducible decreases in relative cell-surface area, with NC-SMCs differentiated using protocol E being the most contractile   and 2 anpercent) B. Althoupercent) B. Stimulpercent) D. We nexpercent) A. We cla NC-SMCs C and shotractile D.Figure 2+ release and contraction of NC-SMCs differentiated from three independent hiPSC lines and compared this with cells derived using protocol B. Intracellular Ca2+ release in NC-SMCs differentiated using either protocol was examined upon stimulation with various vasoconstrictors, such as ET-I (0.1 μM), carbachol (Cch) (100 μM), and angiotensin II (Ang-II) (0.5 μM)  and low to moderate D values of number of events within ROI (D = 0.02–0.17) were obtained when measuring differences between NC-SMCs differentiated using protocols A and B , kinetic parameters of SPs , and number of events within ROI (D = 0.02–0.2)  A. All tht manner B. Low D  A and B C. Compar.02–0.2) D.2+ MP initiated by ET-I, Cch, and Ang-II were observed in NC-SMCs differentiated from the FLB243 hiPSC line , and to a lesser extent NC-SMCs differentiated from the NCRM1 hiPSC line     (Differences in kinetic parameters of Ca11–0.24) E. NC-SMC16–0.24) E. These 07–0.34) E. Import05–0.25) E.D values  (Relative cell-surface area decrease upon stimulation with ET-I (0.1 μM) in NC-SMCs differentiated from three independent hiPSC lines with protocols A or B and showed low 13–0.16) A–S4C. NC13–0.16) A.2+ and contraction assays and compared their responses with HBVP and HBVSMC outputs.Since vSMCs are highly heterogeneous and may differ in developmental origin, we focused on the neural crest population, which has been shown previously to be a major source of vSMCs in the cerebral vasculature. Accordingly, we chose HBVPs and HBVSMCs as a primary human perivascular cell type of neural crest origin as a comparator. We successfully obtained NCCs and derivative NC-SMCs from three independent hiPSC lines using methods based on previously published protocols. We analyzed large ensembles of these NC-SMCs in Ca2+ assay in exhibiting SPs, albeit with slower kinetics of Ca2+ release and reuptake. NC-SMCs differentiated with protocols A and E showed the highest relative cell-surface area decrease upon contraction among all hiPSC-derived NC-SMCs. Although NC-SMCs differentiated with protocol E appeared more similar to HBVSMCs based on Ca2+ release and contractile behavior, their differentiation took much longer and included high concentrations of fetal bovine serum while protocol A was shorter and serum free. Thus, when contractile phenotypes are required, we concluded that protocol A is fit for purpose. On the other hand, NC-SMC differentiation with protocols C and D results in cells functionally closer to HBVPs, although expression of contractile SMC markers was higher compared with HBVPs. Importantly, these assays produced a wide range of detailed information on the vSMC response to stimuli, providing useful information to characterize the hiPSC-derived cells, select the most appropriate protocol, and thus ensure that the vSMCs are fit for purpose in downstream assays.We observed that NC-SMCs differentiated with protocols A, B, or E were most like HBVSMCs in the Ca2+ and contractile responses across NC-SMCs differentiated from different hiPSC lines. This overcomes one major limitation of the efficient use of vSMCs in current studies, namely the lack of specific protein or genetic markers that distinguish perivascular cell types. As many disease states involve switches in contractile phenotype and differential responses to vasoconstrictor stimuli, we expect the methodology to provide a robust approach for quantification and specification of vSMC functionality.In summary, we have provided an unambiguous method for functional analysis of hiPSC-vSMCs that provides multiple parameters to accurately access phenotype in the overall cell population, including heterogeneity in CaFull details are provided in hiPSCs were cultured on Matrigel-coated plates in mTeSR-1 or recombinant vitronectin-coated plates in TeSR-E8 all from STEMCELL Technologies, according to the manufacturer's instructions.2+ release that took place within ∼100 s after administration of the vasoconstrictor. SPs were defined as Ca2+ events after MP if two or more peaks were detected within a given ROI.Images were processed using a freely available plugin “LC Pro” for ImageJ . Free opi is the tracking index number of a single cell; Images were processed using a customized pipeline that included automated cell identification and tracking using CellProfiler. Output data were analyzed using a customized R-based script. Contraction was defined as a percentage of cell-surface area decrease  and 2:(En is total number of bins.All statistical analyses were performed using GraphPad Prism 7 software. The data are reported as mean ± SD. D-metrics was used to measure the difference between two population histograms as a discrete estimator of Kullback-Leibler divergence and calculated using O.V.H. designed and performed research, analyzed and interpreted results, and wrote the manuscript; A.C. analyzed and interpreted results and edited the manuscript; F.E.v.d.H. performed real-time PCRs; C.L.M. designed the research, interpreted results, and edited the manuscript; V.V.O. designed the research, interpreted results, and wrote the manuscript.


O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.Site-specific 2’- Small nucleolar RNAs (snoRNAs) are non-coding RNAs that may function as modification guide RNAs. Specifically, box C/D snoRNAs mediate site-specific post-transcriptional ribose methylation. The C and D boxes are two distinctive short motifs—‘RUGAUGA’ and ‘CUGA’, respectively. They localize close to the ends of a snoRNA molecule and participate in the formation of a specific terminal structure called the kink-turn, or K-turn. This structure consists of a canonical ‘stem I’ formed by 5’ and 3’ termini complementary to each other and a noncanonical ‘stem II’, which contains one U-U and two A-G pairs formed by the C and D boxes. Stems I and II are separated by an asymmetrical 3-nt bulge. Additional slightly diverged copies of the C and D boxes, the so-called C’ and D’ boxes, form an internal stem which is somewhat similar to the terminal one. Single-stranded sequences between the C and D’ boxes and the C’ and D boxes represent regions of complementarity with substrate RNAs targeted for modification. They are usually termed antisense elements (ASEs). The fifth nucleotide upstream of box D or D’ targets the position in the base-paired substrate RNA for modification. The typical structure of a box C/D snoRNA is depicted in O-ribose methylation in eukaryotes [O-methylation activity [Since the discovery of the snoRNA-guided mechanism for 2’-karyotes ,5,6, quikaryotes ,11,12,13activity ,11. BothXenopus oocytes, to further explore box C/D RNA guide activity. This system has already been used successfully for verification of modification guide RNA activities [Drosophila guide RNA for 2’-O-methylation of U2 snRNA at position 28 [Drosophila scaRNA:MeU2-C28 and tested the mutant RNAs for modification activity on U2 snRNA. In general, our data confirm the results of previous studies [One could argue that the RNA-guided modification machinery of higher eukaryotes, especially vertebrates, might have specific features different from those of yeast. Here we use a vertebrate cell system, tivities ,13,14,15ition 28 ,17,18, t studies . MoreoveXenopus laevis females were anesthetized in 0.15% ethyl 3-aminobenzoate methane sulfonate , and a piece of ovary was removed surgically. Oocytes were maintained in OR2 medium [2 medium  for up tDrosophila U2 snRNA and scaRNA:MeU2-C28, both wild type and mutants that lack the CAB-box [Drosophila scaRNA:MeU2-C28 were made by PCR-based mutagenesis and primer annealing-and-extension. Capped Drosophila sense-strand RNAs were transcribed in vitro from linearized plasmids using T3 RNA polymerase. DNA was removed by DNase I treatment followed by DNase I inactivation. RNAs were then purified on MicroSpin G25 columns . The integrity of the purified in vitro-transcribed RNAs was controlled by denaturing RNA gel electrophoresis. Small RNA and low range single-stranded RNA ladders (New England Biolabs) were used to estimate size of the in vitro-transcribed RNAs. Only RNA molecules that migrated on denaturing gels as one sharp band of the expected size with no sign of degradation and/or early termination were used for experiments. RNA concentration was measured with a NanoDrop spectrophotometer .DNA constructs were previously generated to make in vitro-transcribed  CAB-box . AdditioXenopus oocyte system [Xenopus oocytes, followed by 18.4–23 nL (0.5 pmol) of Drosophila U2 snRNA into the cytoplasm. As a control, we injected U2 snRNA alone into the cytoplasm. Oocytes were incubated overnight in OR2 medium at room temperature and nuclei were then manually isolated in 5:1 KCl-NaCl isolation medium [Drosophila U2 snRNA was tested for 2’-O-methylation induced by the injected guide RNAs.We previously described in vitro modification assays using the e system . Brieflyn medium . RNA wasDrosophila U2 snRNA alone or U2 snRNA supplemented with a guide RNA. RNA was extracted after 6–12 h of incubation and analyzed for 2’-O-methylation.In the second type of in vitro modification assay, 150 nuclei were manually isolated and collected in mineral oil. The nuclei were centrifuged to disrupt the nuclear envelope and release the nuclear contents. The resulting nuclear extract was treated with micrococcal nuclease (New England Biolabs) followed by addition of EGTA to inactivate the nuclease. In vitro-transcribed RNAs were added to the nuclease-treated nuclear extract. RNAs were either O-methylation was analyzed by a fluorescent primer extension-based method. We previously described a 6-FAM-labeled oligonucleotide specific for Drosophila U2 snRNA [O-methylation, we performed primer extension with a low concentration of dNTP (0.004 and 0.01 mM) along with control reactions at a high concentration of dNTPs (0.5 mM). AMV reverse transcriptase (New England Biolabs) was chosen to perform 2’-O-methylation analysis. Based on our previous experience [Xenopus oocytes. Fragments were separated on a capillary electrophoresis instrument  using parameters suggested by the manufacturer. To align fragments from different samples we added the Gene Scan-500 Liz Size Standard (Applied Biosystems) to each sample. We determined the exact positions of modified nucleotides with RNA sequencing products. In vitro-trans, cribed Drosophila U2 snRNA was used as a template. For chain termination, one dNTP was mixed with acyNTP (New England Biolabs) at a 1:3 (dNTP:acyNTP) ratio. Data were analyzed and visualized using GeneMapper software (Applied Biosystems).2′-U2 snRNA . To deteperience , this enO-methylation analysis are not truly quantitative and modification efficiency in Xenopus oocytes often depends on the frog used for assays. Therefore, we always injected experimental and control RNAs into oocytes from the same batch and performed enzymatic reactions on control and experimental RNAs simultaneously. For each experiment, we performed several replicates and ran serial dilutions of each sample on capillary columns.Reverse transcription-based methods of 2’-Drosophila Stt3A gene was amplified from genomic DNA and cloned into the pCS2-GFP vector under a CMV promoter. This fragment contains introns 3 and 4, which encode snoRNA:Me28S-U3344a and scaRNA:MeU2-C28, respectively. In addition, two mutant variants were made in which scaRNA:MeU2-C28 was replaced with either an extremely short but still functional single-domain mutant or the same mutant with C in the 2-nt spacer between ASE and box C . The transiently transfected cells were collected after 48 h cultivation and RNA was extracted using the TRIzol reagent. RNA was purified with the Direct-zol RNA MiniPrep Kit .Drosophila snoRNA:Me28S-U3344 and to the 3’ half of Drosophila scaRNA:MeU2-C28. Hybridization was performed at 40 °C for Me28S-U3344 detection and at 34 °C for MeU2-C28 detection in DIG Easy Hyb buffer (Roche). An anti-Dig antibody conjugated with alkaline phosphatase and the chemiluminescent substrate CDP-Star (Roche) were used to visualize the hybridized probes.Total RNA (3 μg per sample) was separated on 8% polyacrylamide–8M urea gels and transferred onto a nylon membrane . The expression of exogenous guide RNAs in HeLa cells was detected with digoxigenin-labeled probes. The probes were specific to O-methylation. For this purpose, we chose scaRNA:MeU2-C28, a Drosophila box C/D guide RNA. Earlier, we showed that this guide RNA is active in the Xenopus oocyte system [The major goal of our experiments was to determine the structural elements in box C/D guide RNAs that play critical roles in RNA-guided post-transcriptional 2’-e system . Here, wDrosophila MeU2-C28 on U2 snRNA was completely eliminated by a point mutation in the ASE at the position targeting U2-C28 for modification  and snoRNA:Me28S-U3344 . We transfected HeLa cells with these constructs and analyzed the expression of Drosophila box C/D snoRNAs by northern blots. We found completely normal processing for both full length and extremely short single-domain MeU2-C28. An extremely short non-functional variant with a CC-spacer between the box C and the ASE (single-domain guide with mutations 9 and 10) accumulated at much lower levels, but otherwise was fully processed (So far, our data confirm 2’-equences . Remarkanstructs A with a rocessed B. From ofunctional eukaryotic box C/D snoRNPs is still challenging, detailed structural analysis of functional and non-functional variants is not feasible. Archaeal box C/D RNP complexes are accessible for the structural analysis. However, a symmetrical two-domain box C/D RNP is essential for function in Archaea [It was previously shown that all four core proteins of box C/D guide RNPs bind normally to a chimeric H/ACA snoRNA that contains only half of the box C/D domain . Here, w Archaea ,26,27,28Our data strongly suggest that there are structural and functional interactions between the terminal and internal K-turn domains of box C/D guide RNAs. When one of the K-turns is mutated, the other K-turn protects the full-length guide RNA from loss of modification activity. At the same time, extremely short single-domain box C/D guide RNAs can also exist and function in the cell, although they are very sensitive to minute changes in their structure. What is the advantage, if any, of having such extremely short and vulnerable guide RNAs? How does a functional guide RNP particle assemble on these very short RNAs? Are there other functions of the short single-domain guide RNAs for which this size is crucial? These and other questions arising from our study still remain open.


Enterovirus C96 (EV-C96) is a newly named type of enterovirus belonging to species C, and the prototype strain (BAN00–10488) was firstly isolated in 2000 from a stool specimen of a patient with acute flaccid paralysis in Bangladesh. In this study, we report the genomic and phenotypic characteristics of two EV-C96 strains isolated from individuals from the Tibet Autonomous Region of China.Human rhabdomyosarcoma (RD), human laryngeal epidermoid carcinoma (HEp-2), and human cervical cancer (Hela) cells were infected with the Tibet EV-C96 strains, and enterovirus RNA in the cell culture was detected with a real time RT-PCR-based enterovirus screening method. The temperature sensitivity of Tibet EV-C96 strains were assayed on a monolayer of RD cells in 24-well plates. Full-length genome sequencing was performed by a ‘primer-walking’ strategy, and the evolutionary history of EV-C96 was studied by maximum likelihood analysis.VP1 sequences, which may represent three genotypes. Phylogenetic trees based on the P2 and P3 regions highlighted the difference between Chinese EV-C96 strains and the EV-C96 prototype strain BAN-10488. All Chinese strains formed a cluster separate from BAN-10488, which clustered with CV-A1/CV-A22/CV-A19.Strain 2005-T49 grew in all three kinds of cells, and it was not temperature sensitive. In contrast, none of the three cells produced CPE for strain 2012-94H. Phylogenetic analysis of the two Tibetan viruses, other EV-C96 strains, and EV-C prototypes showed that EV-C96 strains were grouped into three clusters (Cluster1–3) based on their There is genetic variability between EV-C96 strains which suggest that at least few genetic lineages co-exist and there has been some degree of circulation in different geographical regions for some time. Some recombination events must have occurred during EV-C96 evolution as EV-C96 isolates cluster with different EV-C prototype strains in phylogenetic trees in different genomic regions. However, recombination does not seem to have occurred frequently as EV-C96 isolates from different years and locations appear to cluster together in all genomic regions analysed. These findings expand the understanding of the characterization of EV-C96 and are meaningful for the surveillance of the virus. Picornaviridae, which can be classified to 12 species, including nine enteroviruses, Enterovirus A (EV-A) to EV-H, and EV-J and three rhinoviruses, Rhinovirus A–C [Enteroviruses (EVs) are small, non-enveloped, single-stranded RNA viruses belonging to the family irus A–C . EV-C hairus A–C , 9. GeneVP1 region: EVs that share > 75% nucleotide identity and > 88% amino acid identity in the VP1 region are classified as the same type [The molecular typing method used in recent years to identify different types of enteroviruses is equivalent to the formerly used neutralisation test , 11. Theame type .VP1 sequences, including 11 entire VP1 sequences and 9 complete genome sequences, of EV-C96 strains are available in GenBank, which we used to perform an evolutionary analysis of EV-C96. This study expands the number of EV-C96 full-length genome sequences in GenBank and provides valuable information regarding the molecular epidemiology of EV-C96.EV-C96 is a recently described serotype in the EV-C species based on the molecular typing method  and the Two stool samples were collected in the Tibet Autonomous Region in 2005 and 2012, respectively. One (2005-T49) was collected from a patient with AFP in 2005 in Lazi County in the Rikaze Prefecture of Tibet during the course of poliovirus surveillance in support of the global polio eradication initiative, and the other (2012-94H) was collected from a healthy child in Zhangmu County in Rikaze Prefecture of Tibet during an enterovirus surveillance program of healthy children in 2012.The stool sample (about 2 g) was dissolved in 10 ml of complete phosphate-buffered saline with antibiotics, 1 g of glass beads and 1 ml chloroform, then shaked vigorously for 20 min using a mechanical shaker. Spined for 20 min at 1500 g in a refrigerated centrifuge, and finally aspirated the supernatant for further use. 0.2 ml stool supernatant was inoculated into human rhabdomyosarcoma (RD), human laryngeal epidermoid carcinoma (HEp-2) and human cervical cancer (Hela) cells  and incubated at 36 °C for virus propagation, then the cells were examined for the development of EV-like cytopathic effects (CPE) daily, recorded all observations of inoculated and control cultures for at least 7 days . An EV-B5 cells/ml to all wells in the 24-well plate, then the plates were inoculated with 50 μL undiluted virus stocks. Two different incubators were used: one incubator was adjusted to 36 °C as the optimal temperature for virus propagation, and another one was adjusted to 39.5 °C as the supraoptimal temperature for virus propagation. After adsorption at 36 °C or at 39.5 °C for 1 h, the unadsorbed virus inoculum was removed and 100 μL of maintenance medium  was added to each well. The plates were continually incubated at 36 °C or at 39.5 °C and harvested at 7 time points post-infection  in succession. The 50% cell culture infectious dose (CCID50) was calculated by the Spearmann-Kärber end-point dilution method on monolayer RD cells in 96-well plates at 36 °C. Two Xinjiang EV-B85 strains, HYTY-ARL-AFP02F, which is not temperature sensitive, and HT-LYKH202F, which is temperature sensitive, [For poliovirus vaccine strains, temperature sensitivity usually correlates with attenuation, and vaccine-derived mutant strains which have lost their temperature sensitivity have been shown to be neurovirulent, so temperature sensitivity may be an acceptable method for evaluating the presence of attenuating mutations for enteroviruses . The temnsitive,  were usensitive, .VP1-coding region using the PrimeScript™ One Step RT-PCR Kit Ver.2  with primers E292 and E222 [HEp-2 cell cultures of 2005-T49 (CPE observed) and 2012-94H  were frozen three times to release viruses. For molecular typing, viral RNA was extracted from 140ul HEp-2 cell culture using the QIAamp Viral RNA Mini Kit  and stored at − 80 °C until use. RT-PCR was performed to amplify the and E222 . The RT-The 5′ end sequence of the genome was obtained using a 5′ rapid amplification of cDNA ends (RACE) kit  according to the manufacturer’s instructions. The 3′ end sequence of the genome was obtained by amplification using an oligo-dT primer (primer 7500A) as the downstream primer listed in Table The nucleotide and deduced amino acid sequences of strains 2005-T49 and 2012-94H were compared to those of the prototype EV-C strains by pairwise alignment using MEGA . The evThe full-length genome sequences of the two Tibetan EV-C96 strains described in this study, 2005-T49/XZ/CHN/2005 and 2012-94H/XZ/CHN/2012, were deposited in the GenBank under the respective accession numbers KP984753 and KP984754.Tibetan EV-C96 strain 2005-T49 grew and showed clear CPE in RD cells, HEp-2 cells and Hela cells. In contrast, strain 2012-94H showed no CPE in these three cells. The results were similar when cultured at 33 °C and 36 °C. However, viral RNA of 2012-94H in HEp-2 and Hela cell culture were detected by real-time RT-PCR assay with the cycle threshold (Ct) values of 25.85 and 28.15, respectively, and RD cell culture was real-time RT-PCR negative, suggesting low replication compared with 2005-T49  and indicating a different growth ability in cells for strain 2012-94H  was compared with that of two Xinjiang EV-B85 strains, HYTY-ARL-AFP02F, which is not temperature sensitive, and HT-LYKH202F, which is temperature sensitive . The res3A region, both strains contained a 3-nucleotide deletion at position 5290–5292, and a 2-nucleotide deletion at 7407–7408 in the 3′-UTR. The overall base composition of strain 2005-T49 was 29.82% A, 22.94% G, 22.59% C, and 24.64% U, and that of 2012-94H was 29.99% A, 22.81% G, 22.55% C, and 24.65% U. The polypeptide cleavage sites were predicted based on the sequence of the EV-C96 prototype strain. The nucleotide and amino acid sequence identities in the VP1-coding region between strain 2005-T49 and 2012-94H were 87.3 and 94.4%, respectively, and 2005-T49 and 2012-94H respectively displayed 79.1 and 78.5% nucleotide identity and 93.5 and 92.5% amino acid identity in the VP1 region with the prototype EV-C96 strain.The full-length genome sequences of two Tibetan EV-C96 strains, 2005-T49 and 2012-94H, were obtained. The lengths of the 2005-T49 and 2012-94H genomes were 7471 and 7453 nucleotides, respectively, and both encoded a 2219-amino acid polypeptide. The coding sequences were flanked by non-coding 5′-UTRs of 744 and 726 nucleotides, respectively, and non-coding 3′-UTR of 70 nucleotides each followed by a poly (A) tail composed of a long sequence of adenine nucleotides. The 5′ end sequences obtained from the stool specimens are identical to those obtained from the viral isolates. Alignment of the two Tibetan EV-C96 full-length genomes with the genome of the EV-C96 prototype strain BAN00–10488 showed that they shared the same genomic organisation and same order of genomic regions. However, in the 5′-UTR, one of the Tibet EV-C96 strains, 2005-T94, contained two nucleotide deletions at positions 120 and 139, and strain 2012-94H contained an 18-nucleotide deletion at positions 713–730, which is located in complex RNA elements termed internal ribosome entry sites (IRESs) . In the VP1 region of the two Tibetan EV-C96 strains, all EV-C96 strains in GenBank, and all EV-C prototype strains  contain type I IRES, whereas the cardioviruses and foot-and-mouth disease viruses share another type of IRES, type II IRES. Although sequence identity between different members within type I IRES can be less than 50%, the secondary structure predictions are very similar , 37. It P1 region, which encodes the capsid protein of the virion. In contrast, in the non-structural coding regions P2 and P3, the Tibetan strains showed higher genetic diversity when compared with EV-C prototype strains, including BAN00–10488, which is in agreement with the phylogenetic trees described above. EV-C96 strains from two different countries, China and Finland, showed a close phylogenetic relationship with each other, although there is no clear epidemiological evidence, this could be explained by the fact that the strains from Finland that belong to cluster 1 were thought to be imported from China [VP1 region among the Chinese strains isolated in 2005–2012 indicated that EV-C96 has been circulating in China for many years and has been evolving.The full-length genome characterisation showed that the two Tibetan EV-C96 strains share high nucleotide sequence similarity with the EV-C96 prototype strain BAN00–10488 only in the om China . The genP1 region and the P2 and P3 regions may be a hotspot for recombination events, as exchanges here would not change the viral serotype or affect important functions [P2 and P3 regions of the Chinese EV-C96 strains were highly divergent from those of BAN-10488; in fact, the Chinese EV-C96 strains were divergent from all EV-C prototypes, and strain BAN-10488 was most similar to CV-A1/CV-A22/CV-A19. The difference between the Chinese EV-C96 strains and BAN-10488 was also evident in the phylogenetic trees based on the P2 and P3 regions, in which all Chinese EV-C96 strains formed a separate cluster, and the BAN-10488 clustered with CV-A1/CV-A22/CV-A19.Genetic recombination, an evolutionary pattern observed in most viruses, is a common phenomenon during the evolution of enterovirus , 41. Theunctions . The P2 P1-based tree may represent three different genotypes, and all Chinese EV-C96 strains belong to cluster 1. These findings expand the understanding of the characterization of EV-C96 and are meaningful for the surveillance of the virus.In this study, the full-length genomes of two EV-C96 strains isolated from the Tibet Autonomous Region of China were characterised, and their phylogenetic relationships with other EV-C96 strains and other EV-C serotypes were analysed. The different growth patterns on RD, HEp-2 and Hela cells among EV-C96 strains suggest a unique pattern of phenotypic characterisatics among viruses of this type, one of the reasons leading to this phenotypic character may be due to a deletion of 18 nucleotides in the IRES element in 5′-UTR, which is worthy of further study. There is genetic variability between EV-C96 strains which suggest that at least few genetic lineages co-exist and there has been some degree of circulation in different geographical regions for some time. The three clusters of EV-C96 in the


Bifidobacterium bifidum. This system is based on: i) the broad host range plasmid pWV01, ii) a stress-inducible promoter, and iii) two different signal peptides (SPs) one issued from Lactococcus lactis (SPExp4) and issued from Bifidobacterium longum (SPBL1181). The functionality of BEST system was validated by cloning murine interleukin-10 (IL-10) and establishing the resulting plasmids  in the strain of B. bifidum BS42. We then demonstrated in vitro that recombinant B. bifidum BS42 harboring pBESTBL1181:IL-10 plasmid efficiently secreted IL-10 and that this secretion was significantly higher (sevenfold) than its counterpart B. bifidum BS42 harboring pBESTExp4:IL-10 plasmid. Finally, we validated in vivo that recombinant B. bifidum strains producing IL-10 using BEST system efficiently delivered this cytokine at mucosal surfaces and exhibit beneficial effects in a murine model of low-grade intestinal inflammation.In the last years there has been a growing interest in the use of genetically modified bacteria to deliver molecules of therapeutic interest at mucosal surfaces. Due to the well-recognized probiotic properties of some strains, bifidobacteria represent excellent candidates for the development of live vehicles to produce and deliver heterologous proteins at mucosal surfaces. However, very few studies have considered this genus because of its complexity to be genetically manipulated. In this work, we report the development of a new Bifidobacteria Expression SysTem (BEST) allowing the production of heterologous proteins in  Bifidobacterium breve M16-V, Bifidobacterium longum BB536, and Bifidobacterium bifidum BGN4. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, as defined by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) . Some species of bifidobacteria display beneficial properties for human health and are recognized as GRAS  microorganisms. In addition, some strains are already marketed as food supplements (with probiotic claims) such as ns (FAO) .in situ delivery of proteins of therapeutic interest such as antioxidants, cytokines and protease inhibitors (L. lactis producing either IL-10 or trefoil factor (TTF) to treat, respectively IBD  oral vaccine against Hepatitis C virus (HVC) and Enterovirus 71  anvirus 71 , and (iivirus 71 . Other rvirus 71 .L. lactis is the NICE system (for NIsin Controlled Expression) (nisRK) that hampers its further applications in both fed-batch production and in vivo models. One alternative is the use of an expression system that does not require either the presence of regulatory genes or induction of bacterial cultures before use. Thus, we developed in our laboratory two in vivo stress-inducible expression systems for both lactococci  and lacE system ], which id pWV01 , a dnaK (SPExp4) , and onerent LAB , under Brent LAB . Our resEscherichia coli was grown in Luria-Bertani medium  at 37°C, under aerobic conditions with shaking. L. lactis was grown in M17 medium  supplemented with 1% glucose at 30°C without agitation. Bifidobacteria were cultured in de Man Rogosa Sharpe medium , supplemented prior to inoculation with cysteine , at 37°C under anaerobic conditions  in an anaerobic chamber . Recombinant bacteria were selected by the addition of chloramphenicol (10 mg/L) except for E. coli harboring TOPO plasmid which was selected with kanamycin (50 mg/L).Strains and plasmids used in this work are listed in Table E. coli or L. lactis by electrotransformation (B. bifidum protocols were adapted from Alvarez (600nm) in MRS supplemented with 0.5 M of sucrose. Cultures were then put on ice for 20 min. After centrifugation for 15 min at 4700 rpm, 4°C, pellets were washed three times with cold 0.5 M sucrose + 10% glycerol. This step allowed a 200-fold bifidobacteria concentration. Aliquots were incubated 1 h on ice, and then frozen at -80°C. Cells  were electroporated at 200 Ω, 2.5 kV/cm, 25 μF  with 0.1–0.3 μg of DNA and resuspended in MRS for 3 h at 37°C in anaerobic condition before plating on MRS agar plus chloramphenicol (10 mg/L).General procedures for DNA manipulation were essentially performed as described previously . Plasmidormation  and then Alvarez . BrieflyBifidobacterium sp., we cloned dnaK promoter from a strain of B. longum into pLB141 vector (Table NC_002192). Briefly, a DNA fragment (241 bp) containing dnaK promoter was amplified by PCR from genomic DNA of B. longum (GenBank Accession Number: NC_004307.2) with primers: BglII-PdnaK (5′-CCAAGATCTCAAAAAACBTGAGCCWAMMWYRCTCAACTT-3′) for the coding strand and NheI-PdnaK (5′-AGCTAGCGTTAATAAAGCAAGGTTTATTTTTTTCATAACGTGTGCTCCTTAATTAYTCGTTTGTTCTTACGTTSTTYG-3′) for the complementary strand . The PCR product was then subcloned into a pCR-TOPO plasmid  resulting in pCR-TOPO:dnaK  and cloned into purified backbone isolated from BglII/NheI-cut pLB141 vector, resulting in pLB141:dnaK:Nuc. Nucleotide sequence analysis of this construction revealed a deletion in the SPExp4 (data not shown). To repair this, we performed a directed mutagenesis step by PCR. Briefly, we established pLB141:dnaK:Nuc vector in E. coli TG1 strain in order to purify large quantities of the plasmid, we then performed a first PCR reaction with forward primer: 5′-GTTATGAAAAAAATAAACCTTGCTTTATTAACGCTA-3′ using Pfu turbo DNA polymerase (Promega). Parameters of PCR were a first cycle at 95°C 1 min and 5 cycles at 95°C 30 s, 55°C 1 min, and 68°C 4 min. Afterward, we performed a second PCR reaction on the first PCR product with forward and reverse primers: 5′-TAGCGTTAATAAAGCAAGGTTTATTTTTTTCATAAC-3′). PCR parameters were as follow: first cycle 95°C, 1 min; 20 cycles at 95°C 30 s/55°C 1 min/68°C 4 min; 4°C. Then we performed one digestion with DpnI enzyme  before establishment of the plasmid into L. lactis. The final construction pBESTExp4:Nuc  under the control of PnisA promoter, also known as pLB270 plasmid  and cloned into pBESTExp4:Nuc vector digested with the same enzymes and replacing the corresponding nuc DNA fragment. The resulting vector pBESTExp4:IL-10 was established into B. bifidum.For the expression of IL-10 using BEST system, a DNA fragment encoding for IL-10 mature sequence was obtained from a plasmid harboring murine  plasmid , with NsExp4 (issued from L. lactis) for heterologous protein secretion in bifidobacteria, we replaced this SP with a new one issued from B. longum: SP of the hypothetical protein BL1181 from B. longum NCC2705  of homologous proteins efficiently secreted by bifidobacteria we put a particular interest in this SP since it was one with the highest score of “cleavage site.” For the cloning: a DNA fragment containing the SP of BL1181 (SPBL1181) was PCR amplified from genomic DNA of B. longum strain (GenBank Accession Number: NC_004307.2) with the following primers: EcoRV-SPBL1181 (5′-GGATATCAAGGAGATATAAAAATGACGAACGTACGTGTGATCAAGCCCGC-3′) for the coding strand and NsiI-SPBL1181 (5′-TGATGCATCTGCCTGGGCAGGCTGTGCCGAGCTGAACG-3′) for the complementary strand. The PCR product (132 pb) was subcloned into a pCR-TOPO kit  resulting in pCR-TOPO:BL1181SP (Table BL1181 was obtained from this plasmid with EcoRV/NsiI enzymes (Thermo Scientific) and cloned into purified backbone isolated from blunt-ended-AflIII/NsiI-cut pBESTExp4:IL-10 vector. The resulting plasmid pBESTBL1181:IL-10  and the centrifuged culture resuspended (after one wash step with PBS) into the culture medium: MRS plus cysteine (500 mg/L) in order to apply different stresses: pH4, pH8, 42°C, NaCl (25 g/L) or bile salts . Induction was performed in anaerobic conditions during 30 min. Culture extraction was performed as followings. Protein samples were prepared from 2 ml of induced cultures. After centrifugation , the cell pellet (C) and supernatant (S) were treated separately. The S samples were treated by filtration on 0.25 μm filter then frozen at -20°C. The C fraction was obtained after treatment in a PBS complemented with anti-protease 1× and lysozyme , at 37°C during 1 h and then sonicated (6 × 10 sec with a 30 s of timeout on ice). IL-10 concentration in S and C was assessed by ELISA  and normalized by the final OD600nm.For IL-10 detection, bacteria were grown until exponential phase  during 15 min on ice and then centrifuged. Pellets were resuspended in NaOH. Protein concentration was determined by Bradford assay. Ten micrograms of protein were mixed with the blue gel-loading buffer and loaded in pre-casted gel (Bio-Rad). Proteins were then transferred on PVDF membrane and then blocked overnight in 5% milk in TBST. Membranes were stained with a primary rabbit polyclonal antibody against murine IL-10  and a second goat HRP-conjugated against Rabbit IgG . Blots were reveled using Bio-Rad Kit.Exp4:IL-10 and BS42:BESTBL1181:IL-10 were administered by oral gavage to 6 weeks old IL-10-KO mice during 11 days. To mimic the context of our standard experiment in WT mice   were maintained at the animal care facilities of the National Institute of Agricultural Research . Mice were housed under standard conditions for a minimum of 1 week before experimentation. All experiments were performed in accordance with European Community rules for animal care and were approved by COMETHEA C2EA-45 with authorization n°3445-2016010615159974.9 CFU of bacteria in PBS or PBS alone were intragastrically administered daily for 10 days (gavage period). The inflammation was reactivated 21 days after the first DNBS injection (recovery period) with a second injection of 100 mg/Kg of DNBS solution. Weight loss was monitored daily to assess the severity of colitis. Experiment was performed twice and mice were treated as follow: with ETOH-PBS , with DNBS-PBS , with B. bifidum BS42 wild-type strain , with B. bifidum BS42 harboring pBESTExp4:IL-10 plasmid , and with B. bifidum BS42 harboring pBESTBL1181:IL-10 plasmid .For the induction of colitis, we used an adapted protocol from Martín  based onMacroscopic scores were recorded in the abdominal cavity using previously described system . BrieflyFragment of colon were fixed in Formalin then dehydrated with ethanol 70%, and paraffin-embedded. Section of 3 μm-thick sections were stained with hematoxylin and eosin (H&E). As previously described , colonicColonic contents were homogenized in FastPrep instrument with cold PBS. Supernatants were collected and stored at -80°C until analysis. LCN-2 was measured using R&D ELISA kit  and following manufacturer instruction.6 cells/ml per well and were cultured in 24-well plates at 37°C in a 5% CO2 and 95% air atmosphere. Supernatants were collected after 48 h of culture and were stored at -80°C until further analysis. Splenocytes culture supernatant levels of IL-4, IL-5 , IL-6, IFN-γ (eBioscience), and IL-10  were quantified by ELISA, following manufacturer instructions.The day of sacrifice, spleens were removed and placed in RPMI medium for culture . BrieflyP-value of less than 0.05 was considered significant.Data were analyzed using GraphPad Prism  by non-parametric Kruskal–Wallis test followed by a Mann–Whitney test. A in vivo stress-inducible heterologous protein expression in bifidobacteria, we first cloned the promoter from B. longum dnaK operon  in the gut . Our results show that there was no significant difference in C samples between the two recombinant strains; however, the constitutive production of IL-10 was sevenfold higher in S samples (9283 ± 2981 ng/L/OD) prepared from recombinant BS42:BESTBL1181:IL-10 compared to BS42:BESTExp4:IL-10 strain: S (1377 ± 840 ng/L/OD) (p < 0.05)  or higher at pH8 (p = 0.05). IL-10 amount was much higher in S than in C under pH8 and bile salt stress conditions (p < 0.05). Concerning the analysis in BS42:BESTExp4:IL-10 strain . Finally, the two recombinant strains displayed a different IL-10 secretion profile.Regardless of the stress conditions, IL-10 concentration was higher in S fraction from BS42:BESTn Figure . In contn Figure , nonetheBL1181:IL-10 strain or 50% of mice (4/8) treated with BS42:BESTExp4:IL-10, with a mean level of 0.759 and 0.554 pg/g, respectively  treated with BS42:BESTExp4:IL-10, with a mean level of 0.280 and 0.097 pg/g, respectively  between control and DNBS mice. Our results revealed that IL-10 could be detected in (i) ceacal samples of either 62.5% of mice (5/8) treated with BS42:BESTIn vivo delivery of IL-10 through administration of BS42:BESTBL1181:IL-10 and BS42:BESTExp4:IL-10 strains were then studied in a murine model of low-grade intestinal inflammation in wild type mice  after the second rectal injection and the next 3 days. Mice treated with IL-10-producing bifidobacterial strains tended to lose less weight, 93.06 ± 1.02% and 92.5 ± 1.18% for BS42:BESTBL1181:IL-10 and BS42:BESTExp4:IL-10 groups, respectively, compared to the DNBS-PBS control group, . Nevertheless, only the Ethanol-PBS group is significantly different compared to this colitis control group. The macroscopic score was significantly higher in the DNBS-PBS group compared to Ethanol-PBS group  and BS42:BESTExp4:IL-10 induced a non-significant slight decrease in macroscopic score.p < 0.0001). LCN-2 level was significantly lower with BS42:BESTBL1181:IL-10 group (p < 0.05), and, although not significant, tended to be decreased following treatment with BS42 and BS42:BESTExp4:IL-10.LCN-2, an early marker of inflammation, was measured in colonic content at day 33 Figure . The levBL1181:IL-10 and BS42:BESTExp4:IL-10 groups (respectively p < 0.05 and p < 0.0001) than in the DNBS-PBS group . However, there was no effect on IL-4 and IL-10. The intake of BS42:BESTExp4:IL-10 as well as the BS42 tended to decrease the level of IL-5 and IFN- γ, with no effect on other cytokines.The impact on systemic T-helper cells balance was determined by measuring IL-4, IL-5, IL-6, IFN-γ, and IL-10 cytokines in supernatant from mice splenocytes after 48H of CD3/CD28 re-stimulation  regulated by stress that we validated in B. bifidum. The functionality of BEST system was validated by cloning murine IL-10 cytokine and confirming both its correct expression and biological activity in vivo in a mouse model of low-grade intestinal inflammation. For the expression of IL-10 we confirm that the two recombinant B. bifidum strains were able to produce and secrete (even without any stress) the same IL-10 levels than those previously reported in recombinant bifidobacteria , we observed an IL-10 overproduction reaching 24000 pg/ml. This pH, which could reflect the pH of pancreatic juice when it is spilled into the duodenum, models a condition that Bifidobacterium can shortly encounter following oral administration. It shows the inductivity of our system as, in the large intestine where they are mainly located, Bifidobacterium will encounter a combination of factors and stresses which is difficult to reproduce in vitro. It is also interesting to note that pH8 can also stabilize IL-10. Indeed, it has been shown that at pH8.5, human IL-10 is under a highly stable dimer form  at pH4.5–5 , IL-10 secretion was 2.7-fold increased, confirming the interest of both SPBL1181 and PDnaK promoter. Concentrations of IL-10 in the C fraction were similar for our two recombinant BS42; however, a sevenfold higher concentration was observed in the S of BS42:BESTBL1181:IL-10 compared to that of BS42:BESTExp4:IL-10. SPExp4 allows only a small amount of IL-10 to be secreted compared to SPBL1181. This can be easily understandable by the bifidobacterial origin of this SP compared to SPExp4 which is issued from L. lactis. Thus, BEST system using SPBL1181 seems to be an efficient system to secrete heterologous protein in B. bifidum. In addition, this SP (issued from B. longum) could also work in other Bifidobacterium strains, including B. longum itself; and although preliminary data suggest that SP1181 is functional in B. longum and B. breve, this remains to be confirmed.To improve secretion, we included in the BEST system a new signal peptide issued from in vivo delivery by recombinant bifidobacteria, we used IL-10 KO mice model. In these mice, following oral administration of the recombinant strains, we detected IL-10 in ceacal and colonic content confirming the production of IL-10 in vivo. Of note, these young IL-10 KO mice do not develop colitis despite the administration of DNBS, so we assess the biological efficacy of these recombinant bacteria on a well characterized low-grade intestinal inflammation model  for the same administration period (11 days). If we compare the efficacy between the two recombinant strains, the effects of our BS42:BESTBL1181:IL-10 were more efficient regarding the weight evolution, the macroscopic and histologic scores and LCN-2 levels. In contrast, IL-10-producing L. lactis had a better effect on gut permeability and in cytokines levels. Despite each strain display its intrinsic properties, in the two cases, the presence of IL-10 in the intestine has beneficial effect on the low-grade intestinal inflammation model. It will be interesting to test in a near future a combination of these two recombinant strains producing IL-10 in different murine models of either IBS or IBD. Indeed, the ecological niche of L. lactis is the small intestine whereas for Bifidobacterium it is the colon and we can thus expect a synergic beneficial effect.Several studies have reported beneficial effects of different strains of  colitis . Our B. in vivo IL-10 delivery by B. bifidum. Furthermore, the use of recombinant B. bifidum BS42 secreting IL-10 cytokine in a model of low-grade intestinal inflammation showed its ability to decrease local inflammation and confirmed therefore its potential for delivery of therapeutic molecules in the colon. Despite we consider our expression system is promising, more studies are needed, such as evaluate either daily or weekly bacteria administration which will allow us to determine the advantages of the higher persistency of this genus versus other recombinant bacteria .In conclusion, we succeeded in the construction of a new Bifidobacteria Expression SysTem (BEST) that allowed in vitro characterization of IL-10 production. AM, FC, PR, and A-JW performed the animal experiments. AM performed all dosages. BR read the histological score. SG provided the analytical tools. AM, LB-H, and A-JW interpreted the data and wrote the manuscript. PL and M-JB provided the critical feedback, and helped to shape the research, analysis, and manuscript.LB-H and A-JW designed the study. AF performed the cloning. AM performed the 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.


ACTN3; rs1815739), angiotensin-converting enzyme gene , hypoxia-inducible factor 1 α gene (rs11549465), vascular endothelial growth factor receptor 2 gene (rs1870377), and angiotensin II receptor, type 2 gene (rs11091046), by TaqMan single-nucleotide polymorphism genotyping assays. The proportion of MHC-I was 9.8% lower in men than in women, whereas the proportion of MHC-IIa and MHC-IIx was higher in men than in women . Men with the ACTN3 RR + RX genotype had a 4.8% higher proportion of MHC-IIx than those with the ACTN3 XX genotype. Moreover, men with the ACE ID + DD genotype had a 4.7% higher proportion of MHC-I than those with the ACE II genotype. Furthermore, a combined genotype of ACTN3 R577X and ACE insertion/deletion (I/D) was significantly correlated with the proportion of MHC-I (r = −0.23) and MHC-IIx (r = 0.27) in men. In contrast, no significant correlation was observed between the examined polymorphisms and muscle fiber composition in women. These results suggest that the ACTN3 R577X and ACE I/D polymorphisms independently affect the proportion of human skeletal muscle fibers MHC-I and MHC-IIx in men but not in women.Genetic polymorphisms and sex differences are suggested to affect muscle fiber composition; however, no study has investigated the effects of genetic polymorphisms on muscle fiber composition with respect to sex differences. Therefore, the present study examined the effects of genetic polymorphisms on muscle fiber composition with respect to sex differences in the Japanese population. The present study included 211 healthy Japanese individuals (102 men and 109 women). Muscle biopsies were obtained from the vastus lateralis to determine the proportion of myosin heavy chain (MHC) isoforms . Moreover, we analyzed polymorphisms in α-actinin-3 gene  R577X polymorphism was associated with a higher proportion of myosin heavy chain (MHC)-IIx. The ID + DD genotype of the angiotensin-converting enzyme gene (ACE) insertion/deletion (I/D) polymorphism, in contrast to a previous finding, was associated with a higher proportion of MHC-I in men. In addition, the combined genotype of these polymorphisms was correlated with the proportion of MHC-I and MHC-IIx in men. Thus ACTN3 R577X and ACE I/D polymorphisms influence the muscle fiber composition in Japanese men. Human skeletal muscles are composed of two main fiber types, namely, types I and II; type II muscle fibers are further divided into subgroups IIa and IIx . Type I Genetic factors are suggested to play an important role in determining human skeletal muscle fiber composition. Komi et al.  reportedrs1815739) in the α-actinin-3 gene (ACTN3) (rs4341) in the angiotensin-converting enzyme gene (ACE) (rs11549465) in the hypoxia-inducible factor 1 α gene (HIF1A) (rs1870377) in the vascular endothelial growth factor receptor 2 gene (VEGFR2) (rs11091046) in the angiotensin II receptor, type 2 gene (AGTR2)  , insertine (ACE) , C/T pol (HIF1A) , Q472H ( (AGTR2) , are ass (AGTR2) .Although many studies have shown sex-based differences in muscle fiber composition, conflicting reports are available on the proportion of fast and slow muscle fibers in men and women. Several studies have reported a higher proportion of type I fibers in women than in men , 38–40, ACTN3 R577X, ACE I/D, HIF1A C/T, VEGFR2 Q472H, and AGTR2 C/A, on muscle fiber composition with respect to sex-based differences in the general Japanese population.Therefore, the present study investigated the effects of five previously published genetic polymorphisms, namely, The present study included 211 Japanese subjects. The subjects were recruited from Juntendo University  and Fukuoka University . All of the subjects provided written informed consent before their inclusion in this study. The study protocols were approved by the Ethics Committees of Juntendo University and Fukuoka University.o2peak) was measured using an incremental exercise test (15 W/min for men and 10 W/min for women) on a bicycle ergometer . The test was continued until subjective exhaustion was achieved. We assumed that the participants had reached V̇o2peak when at least two of the following criteria were met: 1) a plateau in V̇o2 with an increase in the work load; 2) blood lactate levels ≥8.0 mM; 3) a respiratory exchange ratio ≥1.15; 4) heart rate within 10 beats of the predicted maximum heart rate; and 5) ratings of perceived exertion ≥19. Respiratory gas analysis was conducted using the mixing chamber method to evaluate the volume of expired air, and the O2 and CO2 fractions were analyzed by mass spectrometry . Lactic acid was analyzed using the portable blood lactate analyzer . To take into account individual differences in body weight, V̇o2 was expressed as kilograms of body weight.Peak oxygen consumption (V̇ACTN3 R577X (rs1815739), ACE C/G (I/D) (rs4341), HIF1A C/T (rs11549465), VEGFR2 Q472H (rs1870377), and AGTR2 C/A (rs11091046) polymorphisms were genotyped using a real-time thermocycler with an end-point analysis mode  by using the TaqMan Single-Nucleotide Polymorphism (SNP) Genotyping Assay . A total of 5 μl of the genotyping mixture, containing 2.5 μl TaqMan GTXpress Master Mix (2×), 0.0625 μl TaqMan SNP Genotyping Assay (40×), and 1.4375 μl distilled water, was mixed with 1 μl genomic DNA (10 ng/μl) for each reaction. Four negative controls were included on each plate. Thermal cycling conditions included an initial denaturation at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s and annealing/extension at 60°C for 20 s. The ACE I/D genotype (rs4340) was determined using the ACE C/G genotype (rs4341), which is in perfect linkage disequilibrium with the I/D genotype as follows: C/C as II, C/G as ID, and G/G as DD , according to the manufacturer’s instructions. The total DNA content was measured using a NanoDrop 8000 spectrophotometer . Subsequently, DNA samples were adjusted to a concentration of 10 ng/μl with Tris-EDTA buffer and were stored at 4°C.  (I/D) rs41, HIF1AFor this, 10–15 mg muscle samples were obtained from the belly of the vastus lateralis under sterile conditions and local anesthesia (1% lidocaine) by using a disposal needle-biopsy instrument . The obtained muscle samples were frozen immediately in liquid nitrogen and were stored at −80°C until further analysis.3VO4, and 1% Triton X-100 or 10 mM HEPES, 70 mM sucrose, 220 mannitol, 100 mM KCl, 2 mM EDTA, 0.1% SDS, and 1% Nonidet P-40] containing a phosphatase inhibitor  and a protease inhibitor . The lysates obtained were centrifuged at 10,000 g for 10 min or 15,000 g for 60 min at 4°C. An insoluble pellet, obtained after homogenization, was suspended in a sufficient volume of SDS sample buffer  and boiled at 95°C for 5 min. MHC composition was determined by performing glycerol SDS-PAGE, according to a method described previously (We assessed myosin heavy chain (MHC) isoforms as markers of the muscle fiber composition , 42, 44.eviously , with sot-test and one-way ANOVA. Independent correlates of each muscle fiber composition were examined by performing multiple linear regression analysis. The combined effect of ACTN3 R577X and ACE I/D polymorphisms was analyzed using Pearson’s correlation coefficient. Statistical significance was set at P < 0.05 for all comparisons. Statistical analyses were performed using JMP Pro version 12 .Shapiro-Wilk test was used to assess the normality of all parameters. Data are expressed as means ± SD. Hardy-Weinberg equilibrium testing was performed for each SNP. Differences in phenotypes between men and women and among groups with different genotypes were analyzed using unpaired o2peak (P < 0.001), whereas the relative proportion of MHC-IIa  and MHC-IIx  was significantly higher in men than in women. The correlations for each subject’s physical characteristics and muscle fiber composition in men and women are shown in r = 0.35, P < 0.001) and MHC-IIa . In women, age was significantly correlated with the proportion of MHC-I , height was significantly associated with the proportion of MHC-IIa , and body mass  and BMI  were significantly associated with the proportion of MHC-IIx.Men and women included in the present study did not show any significant difference with respect to age but showed significant differences in height body mass, body mass index (BMI), and V̇o2peak . The relACTN3 R577X (rs1815739), 209/211 (99.1%) for ACE I/D (rs4340), 207/211 (98.1%) for HIF1A C/T (rs11549465), 208/211 (98.6%) for VEGFR2 Q472H (rs1870377), and 209/211 (99.1%) for AGTR2 C/A (rs11091046). ACE I/D genotype was significantly associated with MHC-I, and the ACTN3 R577X genotype was significantly associated with MHC-IIx. In contrast, no significant association was observed between muscle fiber composition and HIF1A C/T, VEGFR2 Q472H, and AGTR2 C/A polymorphisms. Moreover, no significant association was observed between muscle fiber composition and the analyzed genetic polymorphisms in women. Muscle fiber composition in each polymorphism, i.e., ACTN3 R577X, ACE I/D, HIF1A C/T, VEGFR2 Q472H, and AGTR2 C/A, is shown in ACTN3 RR + RX genotype had a significantly higher proportion of MHC-IIx than men with the ACTN3 XX genotype . Moreover, men with the ACE ID + DD genotype had a significantly higher proportion of MHC-I than men with the ACE II genotype .All of the polymorphisms followed the Hardy-Weinberg equilibrium. The rate of genotyping success was 211/211 (100%) for ACTN3 R577X and ACE I/D polymorphisms on muscle fiber composition in men. In men, the ACTN3 R577X and ACE I/D polymorphisms were significantly correlated with MHC-I  to the stop codon (X) at position 577 (R577X), thus depleting the ACTN3 protein level in fast-twitch skeletal muscle fibers. Previous studies have shown that men with the ACTN3 RR + RX genotype showed superior sprinting performance . Taken together, our results indicate that R-allele carriers  in the ACTN3 R577X polymorphism had a higher proportion of MHC-IIx compared with subjects with the XX genotype in men but not in women.α-Actinins are important structural components of the Z-membrane, and expression of ACTN3 is limited to fast-twitch skeletal muscle fibers. A common nonsense polymorphism in formance , 30. In t et al. , which rACTN3 R577X polymorphism and muscle fiber composition may be associated with the signaling protein calcineurin. Calcineurin is a serine-threonine phosphatase activated by Ca2+-calmodulin, and calcineurin activation plays a key role in the determination and/or adaptation of slow-twitch muscle fibers  Grants-in-Aid for Scientific Research B  and the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities (to Juntendo University and Fukuoka University). H. Kumagai received a Grant-in-Aid for the JSPS Fellow from the JSPS.No conflicts of interest, financial or otherwise, are declared by the authors.N.F. conceived and designed research; H. Kumagai, T. Tobina, N.I-S., R.K., T. Tsuzuki, K.S., E.Y., H. Kumahara, M.A., Y.H., H. Kobayashi, A.K., H.N., H.T., and N.F. performed experiments; H. Kumagai, H.Z., and R.Y. analyzed data; H. Kumagai and N.F. interpreted results of experiments; H. Kumagai prepared figures; H. Kumagai and N.F. drafted manuscript; H. Kumagai, T. Tobina, N.I-S., R.K., H.Z., R.Y., H.N., and N.F. edited and revised manuscript; N.F. approved final version of manuscript.