Salvia miltiorrhiza Bunge. A novel endogenous R2R3-MYB transcription factor, SmMYB36, was identified in this research. This transcript factor can simultaneously influence the content of two types of components in SmMYB36 overexpression hairy roots. SmMYB36 was mainly localized in the nucleus of onion epidermis and it has transactivation activity. The overexpression of SmMYB36 promoted tanshinone accumulation but inhibited phenolic acid and flavonoid biosynthesis in Salvia miltiorrhiza hairy roots. The altered metabolite content was due to changed metabolic flow which was regulated by transcript expression of metabolic pathway genes. The gene transcription levels of the phenylpropanoid general pathway, tyrosine derived pathway, methylerythritol phosphate pathway and downstream tanshinone biosynthetic pathway changed significantly due to the overexpression of SmMYB36. The wide distribution of MYB binding elements  and electrophoretic mobility shift assay results indicated that SmMYB36 may be an effective tool to regulate metabolic flux shifts.Phenolic acids and tanshinones are two major bioactive components in  Salvia miltiorrhiza Bunge, hydrophilic phenolic acids 3 and lipophilic tanshinones 6 are major bioactive components for treating cerebrovascular diseases in clinical studies7. However, the supply of S. miltiorrhiza cannot satisfy the production and application demand due to its long growth cycle and degradation of quality. In recent years, much more attention has been paid to the illustration of metabolic pathways and biosynthetic regulation of secondary metabolites using metabolic engineering or fermentation engineering.In the medical model plant S. miltiorrhiza, which contains a phenylalanine-derived pathway and tyrosine-derived pathway15  is synthesized from L-tyrosine through a tyrosine-derived pathway13. The 4-coumaroyl-CoA and 4-HPLA are combined to generate rosmarinic acid by rosmarinic acid synthase (RAS) and cytochrome P450-dependent monooxygenase (CYP)10. Salvianolic acid B is thought to be a derivative of rosmarinic acid, but more research is needed to clarify the specific process10. The tanshinone biosynthetic pathway covers the cytoplasmic mevalonate (MVA) pathway and plastidial methylerythritol phosphate (MEP) pathway18  and subgroup 5 (VvMYB5b) were likely terpenoid biosynthesis regulators and the R2R3-MYBs of subgroup 3, 4, 5, 6, 7, 13 and 21 were potential regulators of the phenylpropanoid-derived pathway30. The overexpression of PtMYB14 and VvMYB5b influenced the accumulation of terpenoids and phenylpropanoids32, which indicated that members of two above subgroups may regulate both terpenoid and phenylpropanoid biosynthetic pathways. SmMYB36 was grouped with those members by phylogenetic analysis 33, HvMYB1034, DkMYB235, LjTT2c36, PpMYBPA137, OgMYB139, PtMYB13440 and FaMYB1141 belong to subgroup 5 according to their structures, and all function as activators to promote proanthocyanidin accumulation26. AtMYB75 42, AtMYB90 43, PURPLE44, AtMYB11343, GMYB1045, VIMYBA1-346, GmMYB1047, CsRUBY48, PcMYB1049, PyMYB1050, AN252, FaMYB1053, NtAN254 and AtMYB11443, belonging to subgroup 6, could act as activators to regulate anthocyanidin biosynthesis30. Therefore, it is possible to analyse the structure by discussing the function of MYB. It is thought that R2R3-MYB could function individually or cooperate with basic helix-loop-helix (bHLH) and WD-repeat (WDR) transcription factors in regulating metabolite synthesis. For example, the overexpression of either ZmC1 or ZmC1/R promoted tanshinone accumulation in S. miltiorrhiza hairy roots13. The MBW complex (TT2-TT8-TTG1) may cooperatively regulate the production of proanthocyanins56 and flavonoids57, but it is uncertain whether the altered expression of transformed transcription factors led to altered expression of other transcription factors in transgenic lines, which should be studied further.R2R3-MYB factors are the largest family of MYB factors, with 125 members in S. miltiorrhiza due to their transcriptional activation or repression activity on genes of secondary metabolic pathways. The heterologous expression of AtMYB75 (AtPAP1) and snapdragon Rosea1 in S. miltiorrhiza leads to the up-regulation of the expression level of core phenylpropanoid pathway genes and enhanced content of rosmarinic acid and salvianolic acid B59. The overexpression of SmPAP1 promotes the accumulation of rosmarinic acid, salvianolic acid B, total phenolics and total flavonoids in transgenic S. miltiorrhiza Bge.f.alba roots60. Zhang et al. found that SmMYB39 plays a repressor role in gathering rosmarinic acid and salvianolic acid B by inhibiting the gene transcripts of phenolic acid biosynthetic pathway in S. miltiorrhiza8.MYB factors are also applied to manipulate the metabolic process in 32. The accumulation of terpene and anthocyanin was stimulated by the heterogeneous overexpression of PtMYB14 in spruce31. The content of phenylpropanoid-derived compounds and terpenoid compounds were enhanced by the overexpression of AtPAP1 in rose flowers61. The heterogeneous overexpression of ZmC1 or ZmC1/R in S. miltiorrhiza hairy roots could simultaneously mediate the increase in tanshinones and the decrease in phenolic acids by changing the transcript levels of pathway genes in S. miltiorrhiza hairy roots13, which may result from the upward transcript levels of most genes in the MEP pathway and the downstream tanshinone biosynthetic pathway. Heterogeneous and the endogenous transcription factors might exhibit different functions due to different genetic backgrounds or induction effects32.Few studies have been performed on tanshinone metabolic regulation compared to phenolic acid synthetic modulation. The heterogeneous overexpression of VvMYB5b caused an enhancement in carotene and decrease in flavonol and caffeic acid in tomato62. First-level regulation is achieved by structural genes in the biosynthetic pathways and second-level regulation is performed by transcription factors that can control the expression level of structural genes by binding to their promoter regions62. SmPAP1 is able to increase the promoter activity of SmPAL and SmC4H in transiently-transformed tobacco leaves and interact with SmMYC260. ZmC1 can directly interact with the ZmR or the promoter of SmMDC13. MYB-responsive elements  and bHLH binding sites (CANNTG) are widely distributed in the promoter regions of phenolic acid and tanshinone biosynthetic pathway genes in S. miltiorrhiza64. These widespread elements may play crucial roles in regulating metabolic flux shifts.The biosynthesis of secondary metabolites is regulated by a multi-level networkS. miltiorrhiza. This transcription factor could be a potential manipulation tool to control metabolic flux flowing to the tanshinone biosynthetic direction in S. miltiorrhiza.In this research, we found the endogenous R2R3-MYB transcription factor SmMYB36, which could regulate the accumulation of two major secondary metabolites in SmMYB36 had 98% sequence identity with the sequence in the NCBI database (GenBank Number: KF059390.1). Three nucleotide differences were observed at position 52, 171 and 455 , all of which caused amino acid substitutions . Sequence analysis indicated that SmMYB36 contained a complete open reading frame (ORF) and encoded a putative protein of 160 amino acid residues with predicted molecular weight of 18 kDa. Localization prediction results demonstrated that SmMYB36 may localize to the chloroplast, mitochondria and nucleus. The SMART analysis indicated that SmMYB36 contains a complete R2R3 repeat (8 aa to 58 aa and 61 aa to 109 aa) at the N-terminus , subgroup 5 (AtMYB123), subgroup 15  and subgroup 4  were selected for further motif analysis, which aims to discover specific motifs -W-(X19)-W-……-F/I/L/M-(X18)-W-(X18)-W-) of the R2 domain and R3 domain were in accordance with previous reports25. Motif analysis indicated that SmMYB36 has only the DNEI motif, which can be found in subgroup 5 and subgroup 4 , subgroup 4 (C1 motif and C3 motif) and subgroup 15 (WVxxDxFELSxL motif) could not be found  that were already reported or analysed in 60 Fig. . The bidTo reveal the potential function of SmMYB36 in the transcriptional regulation system, the subcellular localization of SmMYB36-GFP was performed in onion epidermis. The GFP fluorescence of the control existed in the nucleus and cytoplasm. The GFP fluorescence of SmMYB36 was intensive in the nucleus and dispersive in the cytoplasm  (from 0 mM to 20 mM) Fig. . HoweverSmMYB36-overexpressing lines were identified. Twelve SmMYB36-overexpressing lines were used in a preliminary experiment to detect the metabolite content  with efficient expression of SmMYB36 and one empty-vector line  were chosen for QPCR analysis. Line EV, line 36-1, line 36-2, line 36-3 and line 36-4 emitted red fluorescence, but no red fluorescence signal was observed in line WT  and SmMYB36 was used to detect the gene integration status of hairy roots  show a decreasing trend of rosmarinic acid, salvianolic acid B, total phenolics and total flavonoids and the increasing tendency of dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone II A  and tyrosine pathway  were down-regulated, except for 4CL1. However, the expression level of RAS1 and CYP98A14 did not change significantly. The expression pattern of phenolic acid biosynthesis pathway genes was consistent with the change in phenolic acid content. The transcript level of methylerythritol phosphate pathway genes  was enhanced substantially, while no obvious expression differences were found in mevalonate pathway genes , except for HMGR2. The tanshinone biosynthetic downstream pathway genes  represented increased expression. The variation in tanshinone biosynthetic pathway gene transcription and tanshinone content was generally consistent. These results show that the main secondary metabolism pathways of S. miltiorrhiza could be regulated by SmMYB36.To further uncover the transcription regulatory function of SmMYB36, the key gene expression level of the phenolic acid biosynthesis pathway and tanshinone biosynthetic pathway was detected using quantitative RT-PCR Fig. . Most caPAL1, C4H1, 4CL1, 4CL2, TAT1, HPPR1, DXS1, DXS2, DXR, MCT, MDS, HDS, CMK, HDR1, GGPPS1, CPS1, KSL1, CYP76AHI, HMGR2) displayed different patterns between SmMYB36-overexpressed and control lines. Among these genes, C4H1, 4CL2, HPPR1, DXR, MCT and GGPPS1 were selected to conduct electrophoretic mobility shift assay (EMSA) due to their promoters contain MYB-binding elements. All of the specific MYB-related probes for these gene promoters GTTA; MBSII, AAAAGTTAGTTA) were synthesized and used for EMSA. Interestingly, SmMYB36 could interact with most of these MYB-related core elements GTTA; MBSII, AAAAGTTAGTTA)  Fig. .Figure 866. The DNEI motif is highly conserved and specific to the proanthocyanidin accumulation66. However, no other motif 75 and dicots 29 provided evidence for the expansion hypothesis of R2R3-MYBs. It is hypothesized that rapid evolution of R2R3-MYBs occurred during the last 400 million years, after evolutionary divergence from bryophytes to tracheophytes75. The amplification of R2R3-MYBs was used to satisfy the specific cellular functions of new plants76, thus adjusting plant metabolism and development through plasticity77. Phylogenetic tree analysis indicates that SmMYB36 is in a close relationship with the AtMYB0, AtMYB23 and AtMYB66 of subgroup 15; AtMYB23 might be an orthologous gene of SmMYB36  and periderm are both specific plant tissues, where secondary metabolites accumulate, indicating the possible regulatory roles of AtMYB23 and SmMYB36 in the development and metabolism of plants. We deduced that SmMYB36 might not only regulate secondary metabolism but also influence plant development; more experimental evidence is needed to clarify.The members of subgroups 4, 5, 6 and 15 were selected to perform motif analysis; these members have relatively close relationships to SmMYB36 in phylogenetic trees Fig. . Motif aB36 Fig. . AtMYB2382. SmMYB36 localized to the nucleus and has transactivation activity, suggesting that it can regulate the transcription of target genes by itself in the nucleus. Some transcription factors function outside the nucleus84. Here, the fluorescence of SmMYB36-GFP is dispersive in the cytoplasm. Cytoplasm is where many physiological processes run. SmMYB36 may be involved in some processes such as transcription in plastids. The function or localization of one transcription factor may be influenced by other transcription factors83. For example, AtMYC1 localized in the cytoplasm and can interact with GL1, leading to the relocalization of GL1 from the nucleus to the cytoplasm and further increasing the number of trichomes83. There may be bHLHs that cooperate with SmMYB36 to regulate the physiological processes in the cytoplasm and nucleus. More research is needed to illustrate the accurate localization results of SmMYB36 using Arabidopsis and tobacco protoplasts.Localization results demonstrated that SmMYB36 is localized in the cytoplasm and nucleus. The function of transcription factors is closely related to their localizationS. miltiorrhiza hairy roots. A negative correlation was found between the content of phenolic acids and the transcription level of SmMYB36. The changes in metabolite content were in accordance with transcript-level changes in metabolic pathway genes. Specifically, the decrease in rosmarinic acid, salvianolic acid B and total phenolic content in transgenic lines were correlated with the reduced transcript expression of PAL1, 4CL2 and TAT1, which suggested the limitation of enzyme activity and substrate content, potentially leading to reduced phenolic acid content. In addition, down-regulation of C4H1 transcription in transgenic lines was also involved in the reduced alteration of total phenolic content. Different 4CLs can direct carbon flux into various phenylpropanoid branch pathways86; 4CL2 is more important than 4CL1 in the phenolic acid biosynthesis of S. miltiorrhiza88. Hence, it is reasonable that 4CL2 was inhibited, but 4CL1 was not affected, in SmMYB36 overexpressed lines. Total flavonoid content of SmMYB36 overexpressed lines decreased remarkably, coordinated with the transcript levels of general phenylpropanoid pathway genes. Overall, the transcript levels of most genes in the upstream phenylpropanoid and tyrosine derived pathways exhibit a decreased tendency compared with control (EV), which might contribute to reduced phenolic compounds in SmMYB36-overexpressed lines. Comprehensively, because of the reduction of metabolic flow to the phenylpropanoid derived pathway and tyrosine derived pathway, the content of phenolic acid was reduced in SmMYB36 overexpressed lines.Over-expression of SmMYB36 can inhibit the biosynthesis of phenolic acids in 26. The R2R3-MYBs of subgroup 4 usually function as repressors participating in phenylpropanoid-derived metabolite accumulation26 and contain the C1 and C3 (EAR or ERF) motif 89. The C3 motif was conserved at the C-terminus of subgroup 4 members and is required for repression activity89. R2R3-MYB, bHLH and WD normally form a functional MBW complex to work. R3-MYBs are the other type of repressors, which can replace R2R3-MYB and competitively bind bHLH proteins to perform repressor activity91. However, SmMYB36 is an R2R3-MYB rather than R3-MYB, and the C3 motif was absent in SmMYB36, which indicates that the inhibition mechanism of SmMYB36 may differ from the above two types repressors and specialize in phenolic acid accumulation. More evidence is needed to determine the inhibition mechanism of SmMYB36.The R2R3-MYBs of subgroup 4 and R3-MYBs are two types of repressors for the phenylpropanoid metabolite pathwayS. miltiorrhiza hairy roots. Changes in metabolite content and changes of pathway gene transcript level were identical. It is generally thought that the MEP pathway, compared to the MVA pathway, plays a larger role in diterpenoid synthesis92. A similar phenomenon was observed in this research: gene expression of the MVA pathway was not obviously altered. However, the contents of dihydrotanshinone I and cryptotanshinone varied from the independent lines  transcription factors93. However, most studies concern the function of R2R3-MYBs in phenylpropanoid metabolism, rather than terpenoid biosynthesis regulation26. This may be because some phenylpropanoid metabolites are closely related to the colour of organisms (e.g. anthocyanin), making them much easier to study94. Second, the regulation of active metabolite biosynthesis is complicated; for example, the complexity of protein complexes (such as MBW complexes and MYB/bHLH complexes95) and the complexity of regulatory networks are difficult to illuminate95.Overexpression of SmMYB36 can promote the biosynthesis of tanshinones in nes Fig. , showingS. miltiorrhiza hairy roots. We found that the phenolic acid content was negatively correlated with tanshinone content in SmMYB36-hairy roots. The altered transcription of SmMYB36 leads to changed expression of biosynthetic pathway genes and modulated the metabolic flux shift from phenolic acid accumulation to tanshinone accumulation. The regulation of metabolic flux has been achieved by the altered expression of secondary metabolic pathway genes24 and transcription factors60 in S. miltiorrhiza. The regulation of transcription factors is thought to function by interacting with cis-elements of secondary metabolic pathway genes. The predicted responsive elements of MYB and bHLH are widely distributed in the gene promoter regions of known phenolic acid and tanshinone biosynthetic pathways64. Our EMSA results indicated that SmMYB36 could interact with most of the predicted core MYB-related elements, providing more evidence for clarifying the metabolic flux shift. These predicted responsive elements and transcription factors may be effective tools to regulate secondary metabolism, which should be studied further. Since AtMYB23 and SmPAP1 can interact with bHLHs (subgroup III f or d + e)96 and SmMYB36 contains a bHLH-binding motif, there may be SmbHLHs that interact with SmMYB36. The illumination of these SmbHLHs will lead to a better understanding of the regulation mechanism of SmMYB36. In addition, transcription factors can regulate primary metabolism. According to our analysis, fatty acid profiles were altered significantly in SmMYB36-hairy roots compared to the control  can be applied to further illustrate the complicated mechanism. SmMYB36 was the only transcription factor found that could regulate the accumulation of two major secondary metabolites at the same time in S. miltiorrhiza. This regulating effect is quite novel, and SmMYB36 is a double-edged sword for the quality of S. miltiorrhiza. Higher expression levels of SmMYB36 promote the accumulation of tanshinones but not phenolic acids. Thus, it is important to control the expression level of SmMYB36 when accumulating the two types of bioactive compounds.Based on the results and analysis, we proposed a model for tanshinone and phenolic acid biosynthesis regulation by SmMYB36 Fig. . SmMYB36S. miltiorrhiza according to the instructions of the RNAprep Pure Plant Kit . The total RNA was reverse transcribed into cDNA using the instructions of the PrimeScript RT Reagent Kit . The whole CDS sequences of SmMYB36 were amplified with primers . The PCR products were recombined into the pDONR207 entry vector using a BP reaction and introduced into the destination vector pK7WG2R or pDEST-GBKT7 using an LR reaction. To generate subcellular localization vectors and a prokaryotic expression vector, the cDNA fragments with digestion sites for SmMYB36 were separately double-digested and cloned into the pA7-GFP vector and pET32a (+) vector. The inserted sequences in the vectors were identified by sequencing .The plasmids pDONR207, pK7WG2R, pK7WG2R-EV and pDEST-GBKT7 were provided by Prof. Cathie Martin . The constructs pA7-GFP and pET32a (+) were kept in our own laboratory. Total RNA was extracted from two-week-old sterile plantlets of SmMYB36 sequences we cloned and the NCBI database. ExPASy, SMART and SOPMA software were employed to predict the molecular weight, domains and secondary structures. Potential positioning prediction was confirmed by cNLS Mapper, Plant-Ploc and TargetP. The amino acid sequence of SmMYB36 was submitted to the R2R3-MYB protein family of A. thaliana, Oryza sativa L., Brachypodium distachyon and Lotus japonicas in the IT3F website (http://jicbio.nbi.ac.uk/IT3F/) to construct the phylogenetic tree  and BLAST tools from NCBI and Phytozome  and the species containing the predicted orthologous genes of SmMYB36 . The hairy roots were harvested after 18 days and used for real-time quantitative PCR analysis, HPLC analysis and a physiological assay.The hairy roots were derived from actin gene with constitutive expression was used as the internal control. The transcript levels of the following genes were quantitated: phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4CL, tyrosine amino transferase (TAT), hydroxyphenylpyruvic acid reductase (HPPR), RAS, CYP98A14, CYP76AH1, acetoacetyl-CoA thiolase(AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), 5-phosphomevalonate kinase (PMK), mevalonate pyrophosphate decarboxylase (MDC), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 2C-methyl-D-erythritol 4-phosphate cytidylyl-transferase (MCT), 4-diphosphocytidyl-2C-methyl-erythritol kinase (CMK), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), GGPPS, CPS and KSL. The primers used in this experiment were used according to previous reports18. Gene expression level was calculated by the ΔΔCT method and represented by their means ± SD. Real-time PCR employed the following protocol: 95 °C for 30 s, 1 cycle; 95 °C for 5 s, 60 °C for 30 s, 40 cycles. The relative expression level of SmMYB36 was shown in Supplementary Fig. The total RNA of hairy roots was extracted according to the instructions of the RNAprep Pure Plant Kit . The total RNA was reverse transcribed into cDNA based on the instructions of the PrimeScript RT Reagent Kit . Real-time quantitative PCR was performed following the instructions of the SYBR Premix Ex Taq II Kit . The The plasmids pA7-GFP-SmMYB36 and pA7-GFP were transiently transformed into onion epidermis using a gene gun . After incubation for 24 hours, the onion epidermis was stained with DAPI  for 20 minutes and washed twice with PBS buffer (pH 7.2). GFP fluorescence, DAPI fluorescence and bright field of onion epidermis were observed under a confocal laser scanning microscope .To determine whether SmMYB36 has transactivation function, the pDEST-GBKT7-SmMYB36 and pDEST-GBKT7 plasmids were transformed into the yeast strain AH109. The 3AT was selected to inhibit transactivation activity (from 0 mM to 20 mM). The transformed yeast cells were first screened on synthetic dropout (SD) medium lacking tryptophan (SD/-Trp/+3AT) and selected on SD medium without tryptophan, histidine and adenine (SD/-Trp/-His/-Ade/+3AT).et al.98 and Zhang et al.8. All the tanshinones and phenolic acids were detected at 270 nm and 288 nm, respectively. All components were determined using a standard curve from Peng et al.99. The same extract of hairy roots used here was exploited for further physiological assays.The hairy roots were dried at 45 °C to a constant weight in an oven. The contents of tanshinones and phenolic acids in hairy roots were determined using HPLC method described by Liang et al.100.The contents of total fatty acids, palmitic acid (C16:0), stearic acid (18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) were determined by GC analysis following the protocol of Li 101. The sum of dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone II A was calculated as total tanshinones in this research. Total phenolics content was determined using the Folin-Ciocalteu method102, with minor modification. Specifically, 0.04 mL extracts, 1 mL distilled water and 1 mL Folin-Ciocaleu reagent were thoroughly mixed. Afterwards, 1.6 mL Na2CO3 (7.5 g/100 mL) was added, and the mixture was incubated in a water bath  for 1.5 hours. The absorbance of samples was measured at 765 nm. Gallic acid was used to construct a calibration curve to determine the total phenolics content. The phenolic acids represent rosmarinic acid and salvianolic acid B. Total flavonoids were detected according to Jia’s method103. The absorption peak was estimated at 506 nm. Using rutin as a standard, the calibration curve was established to determine the total flavonoid content.Photos of hairy roots and their extracts were recorded to display the correlation between colour difference and the total tanshinone contentE. coli BL21. HIS-labelled protein was purified out using Ni-NTA Resin . The elution buffer (pH 8.0) contains 50 mM NaH2PO4·2H2O, 300 mM NaCl and 250 mM imidazole. The promoter fragments were predicted based on the genome sequence of S. miltiorrhiza (http://www.ndctcm.org/shujukujieshao/2015-04-23/27.html) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) databases. The MBS, MRE, MBSI and MBSII specific or core element sequences of promoter fragments were used as probes and the sequences of the same length as the above probes of the SmMYB36 open reading frame were used as control probes. , Least Significance Difference (LSD) and Student-Newman-Keuls (S-N-K). Correlation analysis was performed between the different gene expressions or between gene expression and metabolite content. Pearson test and Spearman test were used to calculate statistically significant correlations . IBM SPSS Statistics was used for various computations.Supplementary information


Chronic infection with hepatitis C virus (HCV) is a major and growing public health concern worldwide, with an estimate of 170 million individuals affected, 3% of the world´s population . With moIn the LAC region, most available data are local and national, rather than regional, and there are some gaps in terms of obtaining more accurate data and effectively preventing and controlling the disease. WHO’s framework for global action and Pan American Health Organization's (PAHO) regional strategy have helped in resolving these issues. There are several estimations on the number of HCV-infected individuals in LAC, some low 2.8–4.6 millions ; intermeIn a systematic review  presentiBetween 2013 and 2014, the first direct acting antivirals (DAAs) were approved in the USA and the European Union. They are more effective, easier to administer, with minimal side effects, and had shown sustained virologic responses over 90% in 12- to 24-week regimens, in monoinfected and coinfected (with HIV) individuals . SeventeDifferent plans for DAAs acquisition have been established in the LAC region. World Hepatitis Day faces us to an unequal world, where amazing progress on anti-HCV treatments obtained during the last 25 years cannot be applied to all, leaving potentially curable patients to a natural history and progression to cirrhosis and hepatocarcinoma, creating large health costs for countries in the near future. We should be aiming at treating all affected, so please, we need that WHO and PAHO continue working in facilitating access to new drugs, that pharmaceutical companies offer the LAC region more affordable drug prices, and most of all, that health authorities and governments invest in their people´s future.Since the first World Hepatitis Day in 2010, WHO, PAHO and many others have pushed for better and comprehensive diagnosis and care for individuals with any type of hepatitis. Despite all the efforts, as of today, the paramount challenges for the LAC region for hepatitis C still are to enhance preventing measures among high-risk populations, to increase awareness among the general population, to facilitate access to diagnosis and hopefully in the near future to augment the number of people treated and cured. It is urgent to stop hepatitis C, as it is curable and any human deserves a chance to be treated. HCV infection should be considered another important priority in the agenda for LAC countries .


This study elucidates the precise role of the RNA‐binding protein Quaking isoform 5 (QKI‐5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA‐binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient‐specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI‐5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI‐5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3′ UTR of STAT3. Importantly, mouse iPS‐ECs overexpressing QKI‐5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI‐5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI‐5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI‐5 is induced during EC differentiation from iPSCs. RNA binding protein QKI‐5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI‐5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs .  Cardiovascular disease is a leading cause of mortality worldwide with pathology being significantly driven by progressive vascular endothelial cell (EC) dysfunction which regulates key pathogenic events such as infiltration of inflammatory cells, vascular smooth muscle proliferation, and platelet aggregation Cell reprogramming is offering new avenues for regenerative medicine with iPSCs proving able to differentiate into nearly all types of cells within the body Vascular cell differentiation is achieved through the coordination of diverse molecular pathways We provide strong evidence that QKI‐5 plays a critical role in the differentiation of ECs derived from iPSCs and acts as a key regulator of CD144 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation through STAT3 signaling. The RNA binding protein QKI‐5 directly binds to 3′ UTR of STAT3 and induced STAT3 mRNA stabilization. Remarkably, QKI‐5 improved neovascularization and blood flow recovery in a hind limb ischemic model. Notably, human iPSCs (hiPSCs) overexpressing QKI‐5 (OE‐QKI‐5) induced angiogenesis in Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice, highlighting, thus, a pivotal role of QKI‐5 in neovascularization, blood flow recovery and angiogenesis.https://www.lgcstandards-atcc.org/?geo_country=gb Millipore, LONZA; England, UK, http://www.lonza.com/ and Thermo Fisher Scientific; England, UK, http://www.thermofisher.com/uk/en/home.html. Antibodies against VE‐cadherin (CD144) (ab33168), PECAM (CD31) (ab28364), Flk‐1 (ab9530), STAT3 (ab119352), pSTAT3 (ab76315), JAK‐1 (ab47435), GAPDH (ab8245), OCT4 (ab19857), eNOS (ab66127), and QKI (ab126742) were purchased from Abcam. Antibodies against vWF (SC‐8068) and QKI (SC‐103851) were purchased from Santa Cruz; Heidelberg, Germany, https://www.scbt.com/scbt/home;jsessionid=jQ9cedHXnegG6a6cJZbpXPfwu6Z4K8PRhaZidcYh8LrI_ccsHWgp!‐97074204#. Antibodies against QKI‐5 (AB 9904) were purchased from Millipore; England, UK, http://www.merckmillipore.com/GB/en?ReferrerURL=http%3A%2F%2Fwww.bing.com%2Fsearch%3Fq%3Dmillipore%26src%3DIE‐TopResult%26FORM%3DIETR02%26conversationid%3D.Cell culture media, serum, and cell culture supplements were purchased from ATCC, England, UK, http://www.sigmaaldrich.com/united-kingdom.html) in Dulbecco's modified Eagle's medium (ATCC) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific), 10 ng/ml recombinant human leukemia inhibitory factor (Millipore); and 0.1 mM 2‐mercaptoethanol (Invitrogen) in a humidified incubator supplemented with 5% CO2. The cells were passaged every 2 days at a ratio of 1:6. Differentiation of iPSCs was induced by seeding the cells on type IV mouse collagen (5 μg/ml)‐coated dishes in differentiation media (DM) containing α‐modified Eagle's medium supplemented with 10% FBS (Invitrogen), 0.05 mM 2‐mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin in the presence of 25 ng/ml VEGF for the time points indicated.Mouse iPSCs were generated in our laboratory using a similar approach as described previously hiPSCs were pre‐differentiated in low attachment plates using StemPro serum free media supplemented with Bone Morphogenetic Protein 4 (BMP4), Activin A, fibroblast growth factor (FGF), and VEGF for 5 days. The pre‐differentiated cells were seeded on fibronectin (Sigma‐Aldrich), while KDR endothelial precursor cells were magnetically sorted on day 6 using MicroBeads Kit (Miltenyi BIotec) and culturing in EGM‐2 media (LONZA) for 3–9 days. QKI‐5 was overexpressed or knockdown by lentiviral gene transfer on day 3 after KDR selection and the cells were harvested on day 3 and subjected to further analysis or labeled for the in vivo Matrigel plug assays. Human umbilical vein ECs (HUVECs) were seeded on collagen I coated plates and cultured in EGM‐2 media. QKI‐5 was overexpressed by lentiviral gene transfer and subjected to further analysis./6 wild type mice.The mouse hind limb ischemia model was performed as described previously t test for two groups or pairwise comparisons or analysis of variance (ANOVA). A value of *, p < .05; *, p < .05; **, p < .01; ***, p < .001 was considered significant.Data are expressed as the mean ± SEM and were analyzed using GraphPad Prism 5 software with a two‐tailed Student's Detailed Methods and Materials are in Supporting Information Appendix, Experimental ProceduresMouse iPSCs were differentiated toward ECs by seeding on collagen IV and DM supplemented with 25 ng/ml VEGF (DM + V) for 2–10 days. The differentiated ECs adopted a typical EC morphology  of differentiated ECs was obtained based on CD144 selection, as it is a robust EC‐specific marker. Based on this pure population of ECs derived from iPSCs, a high expression of QKI‐5 was observed and this was comparable to the EC markers CD144, VEGFA, and the transcription factor STAT3 .Further experiments performed on mouse embryonic stem cells (ESCs) differentiated to ECs, using a similar approach as the iPSCs, verified that QKI‐5 expression was induced in parallel with CD144 expression . These results indicated that QKI‐5 may be implicated in differentiation of ECs derived from mouse iPSC and ESCs. To elucidate the underlying mechanisms regulated by QKI‐5 in EC differentiation, QKI‐5 was overexpressed by lentiviral gene transfer at day 4 of EC differentiation using ECs derived from iPSCs. The cells were harvested on day 6 and subjected to further analysis. QKI‐5 overexpression significantly enhanced expression of the endothelial markers CD144, CD31, eNOS, and VEGFA and the transcription factor STAT3  and lymphatic (Lyve1) markers. Moreover, QKI‐5 induced EC differentiation in an EC‐specific manner, while markers of other cells lineages such as neuronal (Nestin), cardiac (Mef2c), or smooth muscle cell (SMA) were not altered by QKI‐5 overexpression  expression was stabilized in the presence of QKI‐5 , respectively. Moreover, when differentiated ECs were treated with Actinomycin D in a time point experiment from 0 to 24 hours, CD144 expression was stabilized as a decay curve as is shown in Supporting Information Figure S3E. These results demonstrate that QKI‐5 induced the expression and stabilization of CD144 during EC differentiation. Additional experiments have been conducted to address the important question whether QKI‐5 is able to initiate EC differentiation from iPSCs. When QKI‐5 was overexpressed in undifferentiated iPSCs, the cells started expressing the arterial marker Ephrin B2 3 days later, while venous, lymphatic, or markers of other cell lineages, such as Nestin and SMA, were not expressed . Importantly, EC markers were highly induced upon QKI‐5 overexpression in early stages of EC differentiation . Finally, to shed light on the upstream signaling, which activates QKI‐5 expression in early stages of EC differentiation from iPSCs, transcription factor binding analysis on the QKI‐5 promoter has predicted a potential binding site for the transcription factor ETS1. ETS factors, and in particular ETS1 and Etv2, have been reported as key factors that regulate endothelial development Formation of vascular networks is highly dependent on VEGF as a critical regulator of EC differentiation and vasculogenesis during development. Overexpression of QKI‐5 by lentiviral gene transfer induced the secretion of VEGF on day 6 during the EC differentiation process Fig. A. SimilaWhen QKI‐5 expression was suppressed by shRNA during EC differentiation, this resulted in a significant suppression of the EC markers CD144 and CD31  or iPSC ECs OE‐QKI‐5 (iPS‐ECs OE‐QKI‐5) were labeled with Vybrant and injected intramuscularly immediately after induction of hind limb ischemia in mice. PBS was also used as an additional control. After 7 and 14 days, delivery of iPS‐ ECs OE‐QKI‐5 improved neovascularization and promoted significantly higher blood flow in the ischemic limbs compared to the non‐modified, iPS‐ECs controls or PBS controls  in a hind limb ischemia model by showing an enhanced engraftment capacity when compared with non‐modified iPS‐ECs or PBS control groups. Notably, hiPSCs OE‐QKI‐5 induced angiogenesis in Matrigel plug assays in vivo only 7 days after subcutaneously injection in SCID mice, highlighting a clear functional benefit of QKI‐5 in neovascularization, blood flow recovery, and angiogenesis.RNA‐binding proteins add an additional layer of complexity to a series of events, which include processing and splicing of pre‐mRNA, export to the cytoplasm, quality control assessment of mRNA through translation, message decay and stabilization, and translational repression and de‐repression CD144 is a strictly endothelial specific adhesion molecule and is of vital importance in maintaining and controlling the endothelial phenotype through appropriate cell–cell contacts VEGFA is one of the earliest markers for the endothelial lineage during development It has previously been reported that QKI‐5 knockout mice display a range of blood vessel defects during development which result in embryonic lethality This study provides strong evidence that QKI‐5 is capable of inducing differentiation toward ECs and that, in parallel, regulates angiogenesis in both in vitro and in vitro Matrigel plus assays, even in the absence of exogenous VEGF. At the moment, the potential of iPSCs to differentiate toward therapeutic cells is only based on directed empiricism, while they are totally dependent on combinations of growth factors, media, and matrices to favor the desired lineage. In regards to vascular regeneration, it is important to understand the key regulatory pathways such as epigenetic alterations, transcriptional activity, and RNA‐binding patterns associated with the differentiation processes. In particular, although there has been significant progression in the field In summary, our data strongly support the notion that QKI‐5 is induced during EC differentiation, resulting in stabilization of STAT3 expression through direct binding to the 3′ UTR region of STAT3, modulation of VEGFR transcriptional activation and VEGF secretion. Markedly, iPS‐ECs OE‐QKI‐5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia  highlighting a clear functional benefit. This study provides support to the growing consensus that elucidating the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy toward treating cardiovascular disease.A.C. and S.K.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.T., J.B., M.V.‐G., D.D., R.C., C.M., M.E., and Y.H.: collection and/or assembly of data; D.G., A.W.S., L.Z., and Q.X.: provision of study material, final approval of manuscript; A.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, financial support, final approval of manuscript. A.C. and S.K. contributed equally to this article.The authors indicate no potential conflicts of interest.Supplementary Figure S1: QKI is induced in parallel with CD144 in a pure population of differentiated ECs derived from mouse iPS cells. (A‐B) Images taken of iPS‐ECs on days 7 and 10 of differentiation after VE‐cadherin positive selection demonstrate EC morphology, Scale bar, 50 μm. (C) Real time RT‐PCR data on day 10 during the expansion of this pure population of VE‐cadherin positive cells revealed that QKI is highly expressed in parallel with the endothelial marker VE‐cadherin, VEGFA and the transcription factors STAT3, [data are means ± SEM (n=3)] : * p<0.05, ** p<0.01, *** p<0.001.Click here for additional data file.Supplementary Figure S2: QKI is induced in parallel with VE‐cadherin expression during EC differentiation from mouse Embryonic Stem cells (ESC Cells). Further experiments were performed on mouse ESCs differentiated to ECs using a similar approach as the iPS cells. (A) Images of undifferentiated ESCs and (B) 8 days differentiated ESC‐ECs are shown, Scale bar, 50 μm. QKI expression was induced in parallel with CD144 expression during ESC to EC differentiation as real‐time RT‐PCR data revealed , .Click here for additional data file.Supplementary Figure S3: QKI is capable of inducing EC differentiation from iPS cells by stabilising CD144 (A) iPS‐ECs were infected with QKI or an empty vector on day 4 of EC differentiation. Two days later the cells were treated with actinomycin D (1μg/ml) for 6 h or cyclohexamide for 24 hours, western blots show that VE‐Cadherin expression was stabilised In the presence of QKI . (E) When differentiated ECs were treated with Actinomycin D in a time point experiment from 0 to 24 hours CD144 expression was stabilised as a decay curve is shown , .Click here for additional data file.Supplementary Figure S4: QKI is capable of initiating EC differentiation from iPS cells QKI was overexpressed in undifferentiated iPS and 3 days later the cells were harvested, RNA was extracted and analyzed by real time PCR. (A) The data reveal that the cells expressed arterial marker Ephrin B2 but no venous (CoupTF11), lymphatic (Lyve1) EC markers or markers of other lineages such as Nestin and SMA. (B) EC makers were also highly induced after 3 days of QKI overexpression in undifferentiated iPS). (C) ETS1 expression is induced during EC differentiation from iPS cells in a time‐dependent manner, and (D) overexpression of ETS1  via transfection in early EC differentiation from iPS cells leads to the induction of QKI (H), (data are means ± SEM. (n=3), *p<0.05).Click here for additional data file.Supplementary Figure S5: QKI‐5 induced VEGF Receptor 2 (VEGFR2) activation and EC differentiation through STAT3 /JAK1 signaling (A) Luciferase assays show that when STAT3 was knocked down by shRNA on day 3 and next day QKI‐5 was overexpressed the transcriptional activation of the VEGFR2‐mediated by QKI‐5 was ablated. For these experiments QKI‐5 was overexpressed on day 4 and the cells were harvested on day 6 prior to Luciferase analysis. (B) When the differentiated ECs were treated with inhibitors on day 3 of differentiation, to block the JAK‐1 , and STAT3  pathways, QKI‐5 did not activate the EC markers CD31, CD144, eNOS, and FLK‐1 (VEGFR2) and VEGFA at day 6, when overexpressed on day 4. (data are means ± SEM. (n=3), *p<0.05).Click here for additional data file.Supplementary Figure S6: The RNA binding domain (KH Domain) is required for QKI function in EC differentiation (A) Schematic showing QKI structure and localisation of KH domain and experimental design for mutant construct where primers were designed to loop out the domain. When a KH DOMAIN‐mutant QKI construct was overexpressed in the iPS cells (B) QKI no longer induced the expression of the EC markers, and STAT3 signalling (C) .Click here for additional data file.Supplementary Figure S7: Nascent vessels in vivo (matrigel plugs) were stabilised by pericytes/vascular smooth muscle cells in the presence of QKI‐5. Differentiated ECs overexpressing QKI‐5 were subjected to Matrigel plug assays in vivo. Frozen sections from the in vivo Matrigel plugs were stained for Smooth Muscle Actin (SMA) to demonstrate that the nascent vessels in vivo (matrigel plugs) were stabilised by pericytes/vascular smooth muscle cells in the presence of QKI‐5 as smooth muscle alpha‐ actin (SMA) staining is shown, Scale bar, 50 μm.Click here for additional data file.Supplementary Figure S8: QKI significantly improved neovascularization and blood flow recovery in experimental hind limb ischemia. ECs derived from iPS cells (iPS‐ECs), iPS‐ECs overexpressing QKI (iPS‐ECs‐QKI) labeled with Vybrant, or PBS as a control were injected intramuscularly immediately after induction of hind limb ischemia. (A) Sections of adductor muscles were stained with CD31 antibody and capillary density expressed as capillary number per mm2, with (B) further co‐staining with CD31(green) and Vybrant (red) .Click here for additional data file.Supplementary Figure S9: QKI regulates the splicing factor SF3B1 during EC differentiation. Overexpression experiments show that QKI is implicated in the regulation of the splicing factor SF3B1, on day 6 of EC differentiation from iPS cells, as assessed by real‐time RT‐RCR (data are means ± SEM n=3 * p<0.05).Click here for additional data file.Supplementary Figure S10: The splicing factor SF3B1 is induced during iPS cells differentiation. (A) The spicing factor SF3B1 is induced in a time dependent manner during EC differentiation derived from iPS cells as it is shown by real time data,, ).Click here for additional data file.Supplementary Figure S11: Schematic illustration of the identified role of QKI as a key regulator of EC differentiation and angiogenesis. QKI is induced during EC differentiation downstream of ETS1, resulting in stabilisation of VE‐cadherin expression and modulation of VEGFR transcriptional activation and VEGF secretion, through direct binding to the 3' UTR of STAT3, which leads to its activation. iPS‐ECs overexpressing QKI significantly improved angiogenesis, neovascularization and blood flow recovery in experimental hind limb ischemia.Click here for additional data file.Supporting Information 1Click here for additional data file.Supporting Information 2Click here for additional data file.


Purpose. Low-virulence anaerobic bacteria, especially the Propionibacterium acnes (P. acnes), have been thought to be a new pathogeny for a series of disc diseases. However, until now, there has been no histological evidence to confirm this link. The purpose of this study was to confirm the presence of P. acnes in nonpyogenic intervertebral discs via histological observation. Method. Degenerated intervertebral discs were harvested from 76 patients with low back pain and/or sciatica but without any symptoms of discitis or spondylodiscitis. The samples were cultured under anaerobic conditions and then examined using 16S rDNA PCR to screen for P. acnes. Samples found to be positive for P. acnes were stained with hematoxylin-eosin (HE) and modified Brown-Brenn staining and observed under a microscope. Results. Here, 16 intervertebral discs were found to be positive for P. acnes via 16S rDNA PCR and the prevalence was 21.05% (16/76). Among them, 7 samples had visible microbes stained with HE and modified Brown-Brenn staining. Morphological examination showed the bacteria to be Gram-positive and rod-shaped, so they were considered P. acnes. Conclusion. P. acnes is capable of colonizing some degenerated intervertebral discs without causing discitis, and its presence could be further confirmed by histological evidence. Targeting these bacteria may be a promising therapy method for some disc diseases. P. acnes has been found to account for as much as 84% of all isolated bacteria [Since the first isolation of low-virulence anaerobic bacteria, their pathological roles in nonpyogenic intervertebral discs have drawn an increasing amount of attention. Previous studies have demonstrated that the prevalence of low-virulence anaerobic bacterium ranged from 20% to 53% –7. Among P. acnes, had a strong correlation with disc degeneration, Modic changes, sciatica, and low back pain [ P. acnes into normal intervertebral discs of rats or rabbits induced severe disc degeneration and Modic changes [ P. acnes is thought to be involved in a previously undiscovered pathogeny for series of disc diseases.However, epidemiological studies suggested that latent infection of the low-virulence anaerobic bacteria, especially theack pain , 7–9. Foack pain . In addi changes , 12. For P. acnes in intervertebral discs. One rationale is that the P. acnes isolated in these cases may have been contamination rather than original growth because P. acnes is commensal flora existing on skin and it may readily contaminate the intervertebral discs during tissue harvesting [ P. acnes in any intervertebral discs [Nevertheless, some studies have strongly repudiated the idea of a pathological role ofrvesting , 14. Anorvesting –17. For al discs . P. acnes in nonpyogenic intervertebral discs via histological examination. The prevalence of P. acnes was also investigated here. This was the first study to confirm the presence of P. acnes via histological observation, and it provides critical evidence that may dispel this controversy.Generally, the controversy is attributable to the lack of histological evidence. The gold standard diagnostic method, histological identification of bacteria, would not only resolve the question of contamination but also provide undoubtable evidence of its presence in intervertebral discs. For this reason, the primary purpose of this study was to confirm the presence ofA total of 76 patients were included in this study from September 2013 to October 2015. The patients underwent discectomy at single-level lumbar spine due to disc degeneration associated with low back pain and/or sciatica. Patients underwent conservative treatment before surgery. The patients who received antibiotics within the month preceding surgery were not included in this study. Among included patients, the average age was 55.30 ± 14.59 years, and 39 patients were male and 37 patients were female. The levels of surgery were as follows: 2 at L2~3, 8 at L3~4, 41 at L4~5, and 25 at L5~S1. The study was approved by the Institutional Review Board of Shanghai Ruijin Hospital and informed consent forms were provided by every patient.Intervertebral discs were harvested using a routine approach, posterior discectomy with or without interbody fusion. A stringent antiseptic sterile protocol described in a previous study  was used2, 10% CO2, 10% H2) at 37°C for 14 days. To prevent contamination during culture, a pure culture medium without tissue was incubated and examined under the same conditions, serving as a blank control.As in a previous study, all of the samples of intervertebral discs and muscles were immersed in 9 mL tryptone soy broth (TSB) enriched with 10% bovine serum . Tubes c P. acnes. The forward primer was 5′-GGGTTGTAAACCGCTTTCGCCT-3′, and the reverse primer was 5′-GGCACACCCATCTCTGAGCAC-3′. A boil method was used to extract template DNA from culture broth. A positive control  and a negative control (sterile water as template) were included. The PCR was performed as a mixture of 20 μL volume containing 0.8 μL of each primer, 2 μL of 2.5 mM MgCl2, 1.6 μL of dNTP, 2 μL of 10x Ex Taq Buffer, 0.2 μL of 5 U/μL Ex Taq, 2 μL of template DNA, and 10.6 μL of sterilized distilled water . The PCR conditions were as follows: 94°C preheating for 4 min, 94°C denaturation for 45 s, 35 cycles of 94°C denaturation for 45 s, 57°C annealing for 45 s, 72°C extension for 1 min, and a final extension for 7 min at 72°C. Then the amplified product (5 μL) was collected and run on a 1% agarose gel containing 1 μg/ml of ethidium bromide. Electrophoresis was performed in 1x TAE  glacial acetic acid buffer at 120 V. DNA bands were visualized using UV light photography.At the end of the follow-up period, all broth was examined via polymerase chain reaction (PCR) for amplification of 16S rDNA gene. In accordance with the method described by Albert et al.  and our  P. acnes-positive using 16S rDNA was transferred onto Columbia blood agar plate for further bacterial isolation and purification. Briefly, 1 mL of TSB was transferred onto the plates and incubated for 48 h at 37°C under anaerobic conditions . Then, a single colony was selected and transferred onto Columbia blood agar plates to culture for another 24 h.At the end of culture, the broth that had been identified asFor Gram-staining identification, a single colony was selected, transferred into slides, and fixed. The samples were stained with crystal violet for 1 min and washed with water. Then the samples were stained with Gram's iodine for 1 min and washed with water again. The samples were then decolorized with 95% alcohol for 20 s and washed. Finally, the samples were stained with safranin for 1 min and rinsed with water. After staining, each slide was dried in the air and mounted with a coverslip.μm. Routine HE and modified Brown-Brenn staining were performed for each slide. For modified Brown-Brenn staining, the sections were first deparaffinized and rehydrated and then stained with crystal violet for 2 min and then with Lugol's iodine for 2 min. After that, the sections were dipped into acetone for 30 s and washed in water. Then 0.25% basic fuchsin was dropped onto the sections, which were incubated for 5 min. The sections were decolorized with Gallego's solution  for 5 min and then quickly washed twice with acetone. Then the sections were stained with picric acid-acetone mixture (5 mL saturated picric acid and 45 mL acetone) for 30 s and decolorized 3 times with acetone. Finally, the sections were hyalinized with xylene and mounted with coverslips.Intervertebral discs harvested from patients were fixed in 4% formaldehyde for 24 h and then processed with routine paraffin embedding and sectioned at 5 After modified Brown-Brenn staining, the Gram-positive bacteria were stained blue and the Gram-negative bacteria red. All stained samples were observed under a microscope with an oil immersion lens at a magnification of 630x  and digital images were captured at the same time. During the observation, 5 slides were selected for each patient and 10 randomly selected fields per slide were checked. P. acnes in intervertebral discs after anaerobic culture and 16S rDNA PCR examination. However, among the 20 patients, 16 patients  had P. acnes only in intervertebral discs and the culture results of adjacent muscle were negative. Another 4 patients had positive results for P. acnes in both intervertebral discs and adjacent muscles. The samples of adjacent muscles served as a marker of contamination, and positive results in the muscles indicated the presence of contamination associated with the incision during surgery. This suggests that the P. acnes isolated from these 4 patients may stem from contamination during tissue harvesting during surgery while the P. acnes isolated from the other 16 patients was original growth  were found to havel growth . P. acnes. Finally, 53 patients  were absolutely bacteria-free in both discs and adjacent muscles  also showed bacterial growth in the broth during culture, but the results of 16S rDNA PCR suggested that they were not muscles . The P. h colony . P. acnes-positive via 16S rDNA PCR, 15 samples were investigated further via histological examination . None of the patients had any symptoms of discitis before surgery, such as fever or chills. Upon laboratory examination, the white blood cell counts were within normal ranges and there were no indications of infection. Only different degrees of disc degeneration with or without Modic changes were observed in MRI. There were no signs of discitis in MRI, such as osteolytic changes in endplates or vertebrae, pyogenic changes in the discs, or septic changes in the vertebral area surrounding soft tissues. All of 15 P. acnes-positive patients were diagnosed with nonpyogenic degenerated intervertebral discs (Figures Of the 16 intervertebral discs identified as Figures . P. acnes ( P. acnes-positive via 16S rDNA PCR examination, the observed rod-shaped, Gram-positive bacteria were here considered as P. acnes.Of the 15 samples, 7 samples had visible bacteria after HE and modified Brown-Brenn staining, accounting for 9.21% (7/76, Figures P. acnes . In addiP. acnes . ConsideSome bacteria grew in clusters inside the intervertebral discs Figures  and 3, fHere, 15 intervertebral discs found to be bacteria-free using 16S rDNA PCR were randomly selected and examined using the same histological methods. No bacteria were found using either HE or modified Brown-Brenn staining Figures . P. acnes infections at a prevalence of 21.05% as indicated by anaerobic culture and 16S rDNA PCR examination. More importantly, 7 samples were found to have visible bacteria upon histological examination and the bacteria were here considered P. acnes. We concluded that some nonpyogenic degenerated intervertebral discs had latent P. acnes infection, and the bacteria were observable via histology.In this study, nonpyogenic degenerated intervertebral discs were confirmed to have latent P. acnes using immunofluorescence microscopy, but they found that the bacteria colonized as single cells more than in large aggregates so the immune-labeled P. acnes was attributed as contamination [Up to now, direct histological reports of bacteria in nonpyogenic intervertebral discs have been rare. That may be the cause of the vigorous debates surrounding this topic. Stirling et al. were the first authors to report the presence of low-virulence anaerobic bacteria inside intervertebral discs, and they did so via Gram-staining, but the images were not classic or representative . McLorinmination . Recentlmination . P. acnes may be low in intervertebral discs and therefore it was not easy to observe them directly under histology. The study from Stirling et al. suggested that not very many bacteria were in the intervertebral discs [ P. acnes during tissue harvesting, it would produce false-positive culture results of 16S rDNA PCR, but the histological results were still negative. In summary, it is difficult to determine why the 8 samples were culture-positive but histologically negative. More studies are needed to resolve this problem.Here, 16S rDNA PCR served as a primary method of screening method due to its high sensitivity . Histoloal discs . However P. acnes in nonpyogenic intervertebral discs. However, the histological evidence in this study indicated that not all of the isolated P. acnes were contaminated, and at least 7 samples were definitely original growth. First, if the bacteria came from contamination, they could be expected to remain on the surfaces of harvested tissues. In fact, tissue sectioning showed the bacteria to be present within the tissues rather than on the surface. Furthermore, the observed bacteria grew in clusters, further suggesting that they were original growth rather than contamination. Finally, cultures of the adjacent muscles in these 7 patients were negative, indicating that the incision was not contaminated by exogenous P. acnes during surgery and the harvested intervertebral discs had a low possibility of contamination. In this way, the observed bacteria could be considered original growth inside the intervertebral discs.Contamination is the biggest reason to question the presence of P. acnes-induced pyogenic discitis or spondylodiscitis. However, the patients included in this study did not have any signs or symptoms of discitis, and the reasons for this may be complicated. First, the amounts of invaded bacteria may be a key factor in different prognoses. In addition, the different infection pathway by which P. acnes invades intervertebral discs may lead to different clinical outcomes. Conventional viewpoints suggest that P. acnes invades intervertebral discs via iatrogenic operations, which may result in discitis, while other routine modes of infections lead to latent infection [Previous studies have reported some cases ofnfection . Finally P. acnes or other microorganisms in tissues [ Campylobacter pylori in gastric antral biopsies [HE and Brown-Brenn staining were used to visualize the bacteria in this study. Although it is not a routine method of detecting bacteria, HE staining was able to indicate the presence of tissues , 20. Evebiopsies . Brown-Bbiopsies . It has  P. acnes was not conducted in this research. However, due to the diverse phylogenetic formation of P. acnes in intervertebral discs [ Coagulase-negative staphylococci, Corynebacterium propinquum, and Staphylococcus epidermidis, were not examined in this study. A more comprehensive work should be performed to confirm the presence of other bacteria. Finally, more intervertebral samples should be included and investigated via histological observation to confirm the findings of this study.Unfortunately, this study has some specific limitations. First, immunohistochemical labeling ofal discs , it can P. acnes was absolutely confirmed to be present in some nonpyogenic degenerated intervertebral discs, at a prevalence of about 21.05%. This was here confirmed by histological examination. Latent infection of P. acnes may be crucial to many disc diseases, and targeting these bacteria may be a promising therapeutic method.


The Tier 1 Program of the Project P.A.T.H.S.  is a curricular-based program that attempts to promote positive youth development in Hong Kong. In the second year of the Full Implementation Phase, 20 experimental schools  and 23 control schools  participated in a randomized group trial. Analyses of covariance and linear mixed models, controlling for differences between the two groups in terms of Wave 1 pretest scores, personal variables, and random effect of schools, showed that participants in the experimental schools had significantly higher positive youth development levels than did participants in the control schools at post-test, based on different indicators derived from the Chinese Positive Youth Development Scale. The students in the experimental schools also displayed a lower level of delinquency, but better school adjustment than did students in the control schools. Differences between experimental and control participants were also found when students who joined the Tier 1 Program and perceived the program to be beneficial were employed as participants of the experimental schools.


The incidence of community-acquired pneumonia and lower respiratory tract infection rises considerably in later life. Immunoglobulin M (IgM) antibody levels to pneumococcal capsular polysaccharide are known to decrease with age; however, whether levels of IgM antibody to pneumococcal proteins are subject to the same decline has not yet been investigated.This study measured serum levels and binding capacity of IgM antibody specific to the pneumococcal surface protein A (PspA) and an unencapsulated pneumococcal strain in serum isolated from hospital patients aged < 60 and ≥ 60, with and without lower respiratory tract infection. A group of young healthy volunteers was used as a comparator to represent adults at very low risk of pneumococcal pneumonia. IgM serum antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry was performed to assess IgM binding capacity. Linear regression and one-way analysis of variance (ANOVA) tests were used to analyse the results.Levels and binding capacity of IgM antibody to PspA and the unencapsulated pneumococcal strain were unchanged with age.These findings suggest that protein-based pneumococcal vaccines may provide protective immunity in the elderly.NCT01861184), EHPC dose-ranging and reproducibility trial: (ISRCTN85403723).The LRTI trial (LRTI and control groups) was approved by the National Health Service Research Ethics Committee in October 2013 (12/NW/0713). Recruitment opened in January 2013 and was completed in July 2013. Healthy volunteer samples were taken from the EHPC dose-ranging and reproducibility trial, approved by the same Research Ethics Committee in October 2011 (11/NW/0592). Recruitment for this study ran from October 2011 until December 2012. LRTI trial: ( Streptococcus pneumoniae [Community-acquired pneumonia (CAP) has an incidence of 5–11 per 1000 adult population , with aneumoniae . Eighty eumoniae , and thieumoniae , reducedeumoniae , 10 and eumoniae . Polysaceumoniae –16. They. The therapeutic potential of an immunoglobulin preparation enriched with IgM for the treatment of severe CAP is under investigation [The protective role of antibody responses to pneumococcal infection is well described. IgM antibodies are the first class of antibodies to be produced in a primary response to microbial challenge. Although they bind with low affinity they exhibit a broad specificity to a variety of antigens . Murine tigation . Levels tigation –22. LevePneumococcal surface protein A (PspA) is a major and well characterized virulence factor of the pneumococcus. Antibodies directed against PspA augment C3 deposition, and immunization with PspA confers protection to a variety of murine infection models , 24. PspDespite evidence that IgM antibodies may play a role in anti-pneumococcal defence, and that IgG antibodies to pneumococcal proteins have protective potential, the impact of age on levels of IgM antibodies to pneumococcal proteins in humans has not been investigated. This study seeks to address this knowledge gap by investigating changes in levels and function of IgM antibodies against pneumococcal proteins in relation to aging and lower respiratory tract infection (LRTI) using the PspA clade 4 (PspA4) and an unencapsulated pneumococcal strain. It was hypothesised that as subjects age, levels and binding capacity of IgM antibodies against pneumococcal proteins in serum would be impaired and that patients hospitalized with LRTI would have lower IgM levels and functionality when compared to controls.Serum samples used in this study were collected as part of two clinical trials, both of which have already had their primary end-points reported elsewhere: the LRTI trial  and the The LRTI cohort consisted of patients with a diagnosis of LRTI, based on presentation with symptoms of respiratory infection with ≥2 of the following clinical signs: “cough, breathlessness, pleuritic chest pain, fever and increased or new sputum production” . WithoutThe control cohort comprised patients admitted to hospital for reasons other than LRTI. They had no respiratory symptoms, and were age- and gender-matched to the LRTI patients.The healthy cohort was formed of healthy volunteers aged 18–60 with no concurrent co-morbidities.Nasal washes and serum samples were taken from LRTI and control group volunteers within 3 days of hospital admission. Baseline nasal washes and serum samples were taken from healthy volunteers by appointment.lytA qPCR. Urine from LRTI and control volunteers was collected to perform the BinaxNOW  (urine chromatography) test.Nasal washes were processed as described previously , 28 and cpsD (D39-D∆) [An unencapsulated D39 strain with deleted (D39-D∆)  -PBS before adding serial dilutions of serum samples. Standard curves for calculating total IgM were made using purified total IgM from serum  at a level of 1 mg/ml. WCE and anti-PspA4 values were based on a standard pool serum with an IgM level of 4000 arbitrary units/μl .IgM was bound to a goat anti-human IgM polyclonal antibody conjugated to biotin . The conjugate was quantified by streptavidin-alkaline phosphatase  using 4-Nitrophenyl phosphate disodium salt hexahydrate  as the substrate for development. Absorbance at 405 nm wavelength was determined using the Fluostar Omega®  plate reader. A standard curve was fitted using a 4-parameter fit model.Immunoglobulin deposition on whole live unencapsulated pneumococci was assayed using a modification of a published flow cytometry assay . Brieflyp < 0.05.Linear regression analysis was used to correlate levels of antibodies with age. Antibody levels were log transformed to obtain normal distribution then analysed by one-way ANOVA with the Bonferroni correction to compare levels between the three groups. Analyses were performed using GraphPad Prism v5  and significance was set at n = 19) and controls (n = 19). All LRTI patients, and 3 control patients, had been treated with antibiotics before recruitment to this study [Overall, 38 hospital inpatients were enrolled in the LRTI trial, equally divided between patients with a diagnosis of LRTI  of the study’s patients, either by detection of pneumococcal nasal carriage or BinaxNOW (urine immunochromatography) test . Of thes2 = 0.15, p = 0.006)  and PspA4   Fig. , no corr0) Figs.  and c.Fip = 0.01 for ANOVA analysis, p < 0.05 for the difference between LRTI and healthy groups)  or to PspA4  between any of the groups  Fig. . No signps Figs.  and c.FiThe immunoglobulin binding assay was performed to assess changes in the binding capacity of IgM with aging and LRTI status. Sera from ≥60 control group showed no decrease in binding compared to younger volunteers from the healthy group Figs.  and b. CThese results demonstrate that the levels and binding capacity of IgM to pneumococcal proteins are unaltered with aging. Patients with LRTI did not show decreased IgM antibody to D39-DΔ or PspA4 compared to either the control or healthy groups. The study observed higher IgM binding to pneumococcus in a flow cytometry assay for sera from LRTI patients over 60 years.High levels of total IgM in patients with LRTI cannot be an artefact of current infection, but rather of aging, because control patients without infection presented with similar total IgM levels, but the healthy group had significantly lower total IgM levels. Although only IgM antibodies against pneumococcal polysaccharides and proteins can be expected to confer protection against pneumococcal infection, it is nevertheless interesting to note that total IgM antibody levels are maintained despite immune senescence.S. pneumoniae.A study of IgM levels in mice found that total IgM levels were increased in older animals . HoweverIn whole-cell ELISA, in addition to surface protein antigens, cell wall antigens are also present, to which IgM binding may occur; however, whether these antibodies confer meaningful protection has been questioned , 34. It S.pneumoniae in this cohort.Limitations of this study include the small number of samples and the unknown etiology of LRTI of these patients. This study cohort also had significant heterogeneity with respect to many factors affecting immunoglobulin levels; for example, vaccination status, co-morbidities, corticosteroid treatment, and history of previous pneumococcal infection . FurtherIn summary, these findings suggest that IgM antibodies against pneumococcal proteins do not decrease with age and this warrants further investigation into IgM antibodies against pneumococcal proteins and its role in immunity through pneumococcal protein vaccination.