The effects of SP-D polymorphisms on agglutination and other interactions with Mycobacterium bovis bacillus Calmette–Guérin (M. bovis BCG) were evaluated. In comparison with SP-D 92C , our results showed that SP-D 92T  had a lower binding ability to M. bovis BCG, a lower capacity to inhibit phagocytosis, lesser aggregation, poorer survival of bacillus Calmette–Guérin (BCG)-infected MH-S cells, and less inhibition of intracellular growth of M. bovis BCG. The case–control association study showed that the 92T homozygous genotype was a risk factor for TB. However, a lesser effect was seen for polymorphism A538G. In conclusion, the results of functional and genetic analyses of SP-D variants consistently showed that the SP-D 92T variant increased susceptibility to TB, which further confirmed the role of SP-D in pulmonary innate immunity against mycobacterial infection.Surfactant proteins (SPs)-A and -D are C-type lectins of the collectin family and function in the clearance of infectious particles in the lungs. Some polymorphisms of SPs that give rise to amino acid changes have been found to affect their function. Several SP-A gene polymorphisms have been reported to be associated with respiratory infection diseases, such as tuberculosis (TB). However, the relationship between surfactant proteins D (SP-D) polymorphisms and TB is still unclear. To study the associations between SP-D polymorphisms and TB, the correlations of SP-D polymorphisms with TB were examined in a case–control study, which included 364 patients with TB and 177 control subjects. In addition, we cloned two major SP-D exonic polymorphism C92T (rs721917) and A538G (rs2243639) constructs and used these for  Mycobacterium tuberculosis (MTB), is a global public health issue, as it is the leading cause of death among infectious diseases in many countries. Though TB has diminished over a long period of time, it has reappeared and has become a serious concern once again in many regions. The resurgence of TB is also accompanied by increasing numbers of cases of multidrug-resistant TB due to the development of extensively drug-resistant bacterial strains , caused by  strains , 2. A stannually . In Taiwannually . The devannually , 6, genoannually –10, and annually –13, haveannually . In factannually .MTB infection, SP-A mediates the adherence of MTB to human alveolar macrophages and induces mannose receptor activity, which further increases their phagocytic uptake of MTB -A and -D and pattern recognition molecule–mannose-binding lectin/protein (MBL or MBP) . These me of MTB . Surfactocytosis , and the process . Incubatrophages  and limiof cells . Allelicof cells . A studyof cells . Introniof cells , 25. Thuof cells  reportedof cells , 27. SP-of cells . Our preof cells . To the of cells . HoweverMycobacterium bovis bacillus Calmette–Guérin (M. bovis BCG) were investigated. Furthermore, the associations between SP-D functional polymorphisms and susceptibility to TB were also investigated. This work provides evidence that utilizing functional DNA variants of SP-D in clinical association studies, instead of markers of SNPs, advances our understanding of surfactant genetic susceptibility and innate defense mechanisms to pulmonary TB and other lung diseases.In this study, the effects of functional  polymorphisms of SP-D on the interaction between SP-D and A total of 364 patients who were treated for active TB at the General Taoyuan Hospital  and National Cheng Kung University Hospital  between 2010 and 2012 were surveyed consecutively. The inclusion criteria were: adult patients newly diagnosed with active TB, evident lesions of TB on simple X-ray and computed tomography images, and positive results of sputum smears and cultures for mycobacteria. Patients with HIV infection were excluded from this study. The control group comprised 177 volunteer subjects without active TB or a history of TB. Written informed consent was obtained from each patient and volunteer enrolled in this study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committees of Taoyuan General Hospital and National Cheng Kung University Hospital.Genomic DNA was extracted from oral swabs collected from the enrolled TB patients and non-TB subjects using a QIAamp DNA Mini Kit  according to the manufacturer’s instructions. The extracted genomic DNA was analyzed using agarose gel electrophoresis, quantitatively determined by spectrophotometry, and stored at −80°C until use. Genotyping was performed on the two major non-synonymous SNPs of SP-D, rs721917 (C92T) and rs2243639 (A538G), for genetic association analysis. The SNPs were genotyped using the high-throughput, 384-microtiter plate MassARRAY System, SEQUENOM, according to the manufacturer’s protocol. In brief, DNA containing the SNP site of interest was amplified, followed by performance of the homogenous MassEXTEND assay, in which label-free primer extension chemistry was used to generate allele-specific diagnostic products. The allele-specific diagnostic products have a unique molecular weight, and this can be distinguished through the application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry.Hind III and Sac II were obtained from TaKaRa Bio. Dpn I for site mutagenesis was obtained from New England Biolabs. The vector pcDNA3.1/myc-His B was purchased from Invitrogen.QIAprep Miniprep and QIAGEN plasmid Midi Kit for plasmid preparation were obtained from QIAGEN. Restriction enzymes including Escherichia coli XL-1 Blue competent cells (Invitrogen) by the heat shock method, and grown in LB broth at 37°C. Colonies of transformed cells were selected for further gel electrophoresis.The cDNA fragment encoding human SP-D was amplified by PCR using primers  and cloned into HindIII and SacII sites, leading to the formation of recombinant plasmid pcDNA3.1/myc-His B-SP-D (92T/538G). Site-directed mutagenesis was performed using mutagenic primers of SP-D 92T . The recombinant plasmids were then transformed into Hind II and Sac II diagnosis digestion is demonstrated in Figure The construction of recombinant plasmid pcDNA3.1/myc-His B-SP-D 92T/538G) is illustrated in Figure T/538G isFour variants of the rSP-D gene were transfected into human embryonic kidney 293 (HEK 293) cells (ATCC No. CRL-1573) using Lipofectamine LYX™ Reagent (Invitrogen). Culture medium (serum-free Dulbecco’s Modified Eagle Medium) from transfected HEK 293 cells was collected for further western blot analysis. An SP-D ELISA kit (Hycult) was used for final quantification of the four rSP-D variants.l-glutamine, 10 mM HEPES, 10 mM sodium pyruvate, 1% glucose, and 50 µM 2-mercaptoethanol at 37°C in 5% CO2/95% air. M. bovis BCG was cultured on Middlebrook 7H11 agar (BD) at 37°C in an incubator. Bacteria were harvested, and pellets were suspended in PBS plus 10% glycerol. Aliquots  were stored at −80°C. Before infection, the bacteria supplemented with glycerol were replaced with a final concentration of 0.05% v/v Tween 80 and 2 mM CaCl2 PBS to reduce bacillus Calmette–Guérin (BCG) clumping and for subsequent preparation for SP-D binding. To avoid effects on the viability of M. bovis BCG, penicillin and streptomycin were not added to the culture medium of the MH-S cells  that were prepared for infection.The alveolar macrophage cell line derived from BALB/c mice  used in the study was a continuous cell line of murine alveolar macrophages established after transformation of cells obtained by bronchoalveolar lavage from BALB/c mice with simian virus 40. The cells were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 0.05 mg/ml streptomycin, 20 mM 5, 12-well: 5 × 104, 24-well: 2.5 × 104, and 96-well: 5 × 103) were supplemented with 100 mM glucose or 100 mM maltose, with or without 10 mM EDTA, and with one of the four variants of SP-D (0.5 or 1 µg/ml). One hour after infection, un-infected MH-S cells were washed twice with PBS, and the same conditional medium was used for subsequent analysis.A multiplicity of infection (MOI) of 1 (1 bacterium per MH-S cell) was used for all the experiments, with the exception of the phagocytosis assay, in which an MOI of 5 was used. Quantitative bacteria in different cell culture plates  were first dried at 37°C in an incubator for 7 h, and then exposed to ultraviolet light for 2 h to kill any viable bacteria. After blocking with 3% BSA overnight at 4°C, 1 and 10 µg/ml of the four variants of SP-D in PBS with 2 mM CaCl2 were added with or without 10 mM EDTA, and with 100 mM maltose or 100 mM glucose. The results from 1 and 10 µg/ml of the four variants of SP-D were similar, then 1 µg/ml was used to further experiments. After incubation for 1 h at room temperature, the bacterial suspensions were washed and then incubated with a 1:5,000 dilution of anti-SP-D antibody for 2 h at room temperature. After incubation, the suspensions were washed again and then incubated with a 1:3,000 dilution of goat anti-mouse IgG conjugated to HRP for 2 h at room temperature. Finally, TMB substrate was added after washing. Substrate development was stopped after 12 min by 2N H2SO4, and the absorbance of individual wells was determined at O.D. 450 nm using a SpectraMax M2 ELISA reader .Lipoarabinomannan (LAM) from n to MTB . LAM alsn to MTB . Based o9/ml) were labeled by incubation with FITC (Sigma-Aldrich) at 1 µg/ml in PBS for 2 h at 37°C. Thereafter, FITC-labeled bacteria were washed twice with PBS, then opsonized, and used for infection as described above. Macrophages infected by FITC-labeled M. bovis BCG were analyzed and gated using flow cytometry to assess the effects of the four rSP-D variants on the phagocytosis of MH-S cells. Briefly, MH-S cells (105) were incubated with FITC-labeled M. bovis BCG (5 × 105 CFU) in a 6-well cell culture plate  in the absence or presence of the four SP-D variants at concentrations ranging from 0.5 to 1 µg/ml. After incubation for 1 h, MH-S cells were scraped out after fixation with 4% formaldehyde PBS (pH 7.2) and examined using a flow cytometer  equipped with an argon laser that emits at 488 nm. At least 1 × 104 of cells with FITC-labeled bacteria were counted in the FACS analysis.A Vybrant Phagocytosis Assay Kit  was used to measure the phagocytosis of mycobacteria by MH-S cells. Briefly, bacteria (10R × D2)/4, where R = ratio of a circle’s circumference to its diameter and D = diameter. To determine the effects of the four variants of SP-D on macrophage cell migration, 2.5 × 104 MH-S cells were seeded in a 24-well cell culture plate and infected with M. bovis BCG as described above. The culture medium was harvested 2 days after infection and added into a new 24-well cell culture plate equipped with Millicell Hanging Cell Culture Inserts . Each insert was seeded with 5 × 104 MH-S cells. After 1 day of culture, the inserts were removed and observed using an Olympus IX-71 inverted microscope to count the cells migrated through the membrane. The chemoattractive MH-S cells were quantified using ImageJ 1.39g software.To further characterize the effects of the four SP-D variants on the agglutination of MH-S cells after mycobacterial infection as described above, micrographs were taken 2 days after infection using an Olympus IX-71 inverted microscope. The area of the agglutinates was measured using ImageJ 1.39g software . The diameter was estimated from the area based on the circular form of the aggregates according to the formula Area = (3 MH-S/100 μl medium) were infected with M. bovis BCG as described above. Two days after infection, surviving MH-S cells were evaluated using a Cell Counting Kit-8 (CCK-8)  with a SpectraMax M2 ELISA reader (Molecular Devices).Quantitative bacteria in a 96-well cell culture plate (5 × 104 in a 12-well cell culture plate). Aliquots of MH-S cell lysate were then plated onto 7H11 agar (BD) to quantify the intracellular growth of viable M. bovis BCG. Colony-forming units were counted after 28 days of bacteria culture.To determine the effects of the four SP-D variants on the intracellular viability of bacteria after phagocytosis by macrophages, 100 µl RIPA buffer  were added to the culture plate 2 days after MH-S cells had been infected . In the genetic association study, the quality of the genotype data was evaluated by Hardy–Weinberg equilibrium proportion tests. χ2 tests were used for association analyses. Odds ratios and 95% confidence intervals were calculated from contingency tables. SNPs showing significant associations  in the tests were further evaluated using logistic regressions adjusted for age and gender in odds ratio analysis. Statistical analyses for the association study were performed using SPSS 17.0 software .Results are given as means ± SEs. ANOVA was used to compare the rSP-Ds. All P < 0.0001). There was no significant difference between the groups regarding age (P > 0.05).Surfactant proteins D genetic polymorphisms are most often associated with pulmonary diseases, and our hypostatized genetic variants of SP-D may have different levels of contribution to the pathogenesis of TB. Therefore, we analyzed the genotype and allele frequencies of SP-D in TB- and non-TB-infected patients. 364 case subjects with diagnosed TB and 177 control subjects without a history of TB infection were enrolled. The mean age of the cases and controls was 55.22 and 57.75 years, respectively (Table P = 0.050), and this significance was reduced after adjusting for age and gender  and A538G (SNP ID: rs2243639) could not be derived from the Hardy–Weinberg principle. The association between TB and rs721917 genotypes was marginally significant (M. bovis BCG in vitro (SP-D binds to the surface of pathogens as a first line of defense to prevent pathogens binding before TB infection). We analyzed the binding activities of the four genetic variants of rSP-D with M. bovis BCG and found that all four genetic variants of rSP-D bound to M. bovis BCG in a dose-dependent and specific manner that could be inhibited by 10 mM EDTA or 100 mM maltose, but not by 100 mM glucose  and rSP-D (92T/538G)]  , indicating that the avidity of SP-D binding to M. bovis is affected by the change in amino acid at residue 11. By contrast, the change in amino acid residue 160 (from Thr to Ala) did not lead to a significant difference in binding ability to M. bovis BCG.To confirm that SP-D 11Thr/160Thr  confer greater protection against TB infection as per the genetic analysis, as well as to further investigate the protective effects of four genetic variants of rSP-D in the progression of TB infection to TB disease, we constructed four genetic variants of SP-D, and then, respectively, measured their protective ability against  Figures A,B. At pP-D 92T/58A and rSM. bovis BCG infection, but with different potencies. As the results presented in Figure All rSP-D variants inhibited phagocytosis by alveolar macro_phages following Aggregation of infected MH-S cells, an event in which a collection of macrophages is formed during mycobacterial infection, was also evaluated. The rSP-D variant 11Thr/160Thr (92C/538A) inhibited the aggregation of infected MH-S cells more obviously than the other variants Figure B. The prWe further analyzed the associations between aggregation of MH-S cells and various rSP-D mutants through a chemotaxis assay. However, the recruitment of nearby MH-S cells after mycobacterium infection did not differ between the four rSP-D variants Figure C.M. bovis BCG, a cell survival assay was performed using CCK-8. All variants of rSP-D improved the survival of infected MH-S cells in a concentration-dependent manner  pathogenesis. Aliquots of infected MH-S cell lysate were plated onto 7H11 agar to evaluate the effects of the four rSP-D variants on intracellular bacteria growth. Our results showed that all four variants of rSP-D inhibited intracellular h Figure B. The adG Figure B. There M. bovis BCG, a good surrogate representing MTB, to study the role of SP-D polymorphism in mycobacterial infection and the connection with TB. Our results showed that M. bovis BCG interacted with the different rSP-D variants at various levels; these effects were abolished following the addition of maltose or EDTA, which causes a competitive effect with the lectin domain of SP-D or depletion of calcium. At first glance, it appeared that interaction of SP-D with MTB was dependent on its carbohydrate recognition domain (CRD). However, a closer look at the comparison of innate immunity against TB infection of genetic variants in the N-terminal and collagenous regions of rSP-D showed different degrees of activity. We found that SP-D residue 11 polymorphism, Thr (92C) and Met (92T), affected the binding activity of SP-D to M. bovis BCG, phagocytosis of infected MH-S cells, aggregation of infected MH-S cells, cell death of infected MH-S cells, and intracellular growth of M. bovis BCG. By contrast, the effects of SP-D 160 polymorphism, Ala (538G) and Thr (538A), were weaker than those of residue 11 polymorphism, and did not appear distinctly. From the results of SP-D variant functional and genetic analysis, residue 11 Met (92T) appeared likely to cause susceptibility to TB, which was validated in our TB-infected patients.In this study, we used in vitro and in vivo  are also found within the SP-D gene , 32; mos in vivo , indicatM. bovis BCG, residue 160 Ala (92C/538G) showed a lesser binding activity than 160 Thr (92C/538A) when residue 11 was Thr, but not when it was Met. The SP-D variant 11M/160A (92T/538G) showed weaker inhibition of phagocytosis than the other variants at an SP-D concentration of 1 µg/ml. Our results implied that polymorphism A539G (160 residue Ala/Thr) has less impact than C92T (11 residue Thr/Met) and may exhibit an effect only under specific conditions or through interaction with other genetic factors. The protective effect of homozygous 538A (160 residue Thr) against TB was consistent with the results of functional studies mentioned above.Another coding single-nucleotide variation is located on exon 4, and the collagen-like region in the mature protein is rs2243639, known as A538G and SPD A160. As with other genetic alleles, the two-marker SP-D/SP-A haplotype, DA160_A/SP-A2 1A, is protective against the development of neonatal respiratory distress syndrome . SP-A2/SSurfactant proteins D binds to the bacterial surface and reduces the uptake of bacteria by alveolar macrophages . Also, Sin vitro. Therefore, it is likely that SP-D polymorphisms C92T and A538G affect susceptibility to TB, which is in agreement with our findings in an association study of clinical TB patients. There were some limitations to our study. First, we were not able to differentiate whether or not these genetic variants of rSP-D undergo multimerization (HMW vs LMW) under our experimental conditions. HMW SP-D multimers are only partly dependent on disulfide crosslinking of the N terminal region, and some SP-D subunits are non-covalently associated depending on the protein concentration in the solution (in vivo effects of the genetic variants of rSP-D in a TB-infected animal model due to a small yield of recombinant full-length human SP-D from HEK 293 cells. Third, although high antigenic and structural similarities exist between MTB and M. bovis BCG, use of M. bovis might not wholly represent the infectious nature of MTB in hosts. MTB could not be utilized in this study due to a lack of availability of the necessary safety equipment in our laboratory.In this study, we demonstrated that the genetic variants of rSP-D in the N terminal and collagenous region confer different degrees of innate immunity against TB infection solution . Severalsolution , 37, 43.in vivo and in lung diseases, wild-type and transgenic Sftpd−/− mice expressing either the human SP-D Met11 or Thr11 allelic variants were generated (in vivo and in vitro studies, which implies that the homeostatic and anti-microbial effects of SP-D are primarily mediated by its CRD region (However, to evaluate the influences of SP-D polymorphisms enerated –46. In aenerated , 45. In D region , 48.MTB, this study elucidates that it is also influenced by the genetic variants in the N-terminal and collagenous domains of SP-D. The two major SP-D polymorphisms C92T and A538G, and especially C92T, appeared to affect susceptibility to TB. Our association study of the susceptibility to TB infection in patients also supported this hypothesis.In conclusion, our results demonstrated the role of SP-D in innate immunity against TB infection. Although this mostly depends on the binding of CRD with The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committees of Taoyuan General Hospital and National Cheng Kung University Hospital.M-HH, C-YO, W-YH, J-YW, and LW contributed to the conception and design of the study; M-HH and W-YH performed experiments; C-YO and H-FK analyzed the data; C-YO and S-WL collected TB patients and performed genetic analysis; LW and J-YW wrote the first draft of the manuscript; and all authors contributed to manuscript revision, and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the  Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.The nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast  Sacchrevisiae . The tworevisiae –12. The revisiae –16. Addirevisiae –19. Theirevisiae . The hetrevisiae ,20,21. Rrevisiae ,20. Rpa4revisiae ,22 and irevisiae ,23. Finarevisiae ,24.in vivo  for 30 minutes. Plugs were stored at 4°C till usage in TE or 0.05 M EDTA [pH 8.0]. Plugs were loaded on 0.8% agarose gel in 1×TAE buffer  and run in 1×TAE buffer for 50h in CHEF-DRIII System with chiller unit (Bio-Rad) with following parameters: 3V/cm, initial S/time 250 sec, final S/time 900 sec, angle 120. Gel was stained with ethidium bromide (10 mg/ml in water). Gel was processed for Southern blotting: DNA was transferred on nylon membrane by passive transfer in 10×SSC buffer . Membranes were UV-cross-linked and hybridized overnight with 32P-labeled single-stranded DNA probes at 42°C. Blots were washed twice with 2×SSC and 1% SDS for 20 min and once with 0.5×SSC and 1% SDS for 20 min at 37°C. rDNA locus (chromosome XII) was detected with 18S specific probe (CATGGCTTAATCTTTGAGAC). This protocol was kindly provided by B. Pardo , and is extensively described in [PFGE is a technique that resolves chromosome-sized DNA molecules in an agarose gel. Over-night YPD cultures at 30°C were harvested in 0.1% azide Na and kept on ice. Cells were washed twice in cold 0.05 M EDTA [pH 8.0]. ~2.10ribed in .TRO was performed as previously described ,43. SlotS1 Fig(A) Schematic representation of GIM interaction assay. Enrichment ratios between control and SGR are used as read-out to map genetic interactions. (B) Relative enrichment of each barcode (black cross) along chromosome (in red) is used to map genetic linkage. Green curve represents mean in a sliding windows of 20 barcodes. Local maximum in such curve was used to identify locus of interest: RPA49 as positive control ; RPA135 in SGR1 ; RPA190 in SGR2 .(TIF)Click here for additional data file.S2 FigS. cerevisiae (NP_015335.1), compared with S. pombe (NP_595819.2), H. sapiens (NP_061887.2), M. musculus (NP_033112.2), D. rerio (NP_956812.2), D. melanogaster (NP_476708.1) and C. elegans (NP_492476.1). Color code from light to dark blue indicates residue conservations. Mutated residues at position 252 (Y to H), 299 (D to G), 300 (S to F), 301 (F to S or L) and 305 (R to L) are depicted in red.(A) Structural domains of Rpa135 are depicted ,5, inclu(TIF)Click here for additional data file.S3 Figrpa49ΔCt/RPA135-F301S; rpa49ΔCt; RPA135-F301S and RPA49) were separated on the agarose gel and analysed by Southern blot using rDNA-specific probe.Chromosomes from indicated strains (same as in (TIF)Click here for additional data file.S4 Figin vitro transcription assays of Pol A* (lacking Rpa34 and Rpa49) and Pol A* bearing Rpa135-F301S are complemented with recombinant A34.5/A49 heterodimer. Promoter-dependent assays were performed as in Promoter dependent (TIF)Click here for additional data file.S5 FigWT, RPA135-F301S, rrp6Δ and RPA135-F301S -rrp6Δ grown at 25°C for 3 days.Ten-fold serial dilutions of (TIF)Click here for additional data file.S6 FigTailed template and promoter-dependent assays were performed as in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 MovieThe movie starts with the closed cleft conformation (PDB 5W66 ), in whi(MP4)Click here for additional data file.


Methods of generating and expanding AT2 cells derived from induced pluripotent stem cells (iPSCs) have been established and are expected to be applicable to disease modeling. Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by dysfunction of lysosome-related organelles, such as lamellar bodies (LBs), in AT2 cells. From an HPS type 2 (HPS2) patient, we established disease-specific iPSCs (HPS2-iPSCs) and their gene-corrected counterparts. By live cell imaging, the LB dynamics were visualized and altered distribution, enlargement, and impaired secretion of LBs were demonstrated in HPS2-iPSC-derived AT2 cells. These findings provide insight into the AT2 dysfunction in HPS patients and support the potential use of human iPSC-derived AT2 cells for future research on alveolar lung diseases.It has been challenging to generate   •HPS2-iPSCs and cHPS2-iPSCs were generated from HPS2 patient fibroblasts•Anti-NaPi2b antibody was useful for isolating AT2 cells from human lung and AOs•The enlargement and abnormal distribution of LBs were observed in HPS2-AOs•Impaired surfactant secretion was demonstrated in HPS2-AOs Pulmonary surfactant secretion by alveolar type 2 (AT2) cells is crucial for alveolar homeostasis. Gotoh and colleagues generated Hermansky-Pudlak syndrome type 2 (HPS2) patient-derived iPSCs and their gene-corrected counterparts and differentiated them into alveolar organoids (AOs). In HPS2-AOs, aberrant lamellar bodies and impaired surfactant secretion were demonstrated. Human iPSC-derived AOs might be useful for investigating alveolar lung diseases. Alveolar type 2 (AT2) cells are tissue stem cells that maintain homeostasis of the alveolar region of the lung . They seAP3B1 gene, which encodes the β3A subunit of the AP-3 complex, which is involved in intracellular membrane traffic. It was previously reported that approximately 40% of HPS2 patients had PF and that 78% of HPS2 patients with PF were children (Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive hereditary disease caused by mutations in the genes involved in the formation and maturation of LROs and characterized by oculocutaneous albinism and bleeding diathesis . Among tchildren .In this study, we generated HPS2 patient-derived iPSCs (HPS2-iPSCs) and gene-corrected iPSCs (cHPS2-iPSCs) and differentiated them into AOs . Based on the comparison of these AOs, we report the AT2 cell dysfunction of HPS2-AOs.AP3B1 gene and he was histologically diagnosed with nonspecific interstitial pneumonitis at 20 months of age  A. The HPs of age   and CD47 combined with or without CD26 . The expression levels of representative AT2 markers  and alveolar type 1 (AT1) markers (AQP5 and AGER) were not significantly different among the AOs derived from control hPSCs and HPS2-iPSCs and cHPS2-iPSCs  showed no differences between HPS2-AOs and cHPS2-AOs (Ap3b1 mutant (Pearl) mice did not develop PF spontaneously , a fluorescent probe used for visualizing LBs . InteresHPS2-AOs A–3C. ThiHPS2-AOs A and S3Fn P2-AOs A and 3B.n P2-AOs , was locd to LBs D. In eled to LBs E. Furthed to LBs G, consisd to LBs . We ther+ cells of both AOs were reseeded on coverglass chambers, and live cell imaging was performed after staining with LT and FM1-43, a fluorescent probe that labels free lipids and which has been used to evaluate the exocytosis of LBs from primary AT2 cells  , we evalT2 cells   turned yellow-green due to the fluorescence of FM1-43 (green) in the cHPS2-AO(P0) epithelium, whereas the color of LBs showed no change in the HPS2-AO(P0) epithelium. Scale bars, 5 μm.AP3B1 mRNA is very low in HPS2-iPSCs due to NMD, and the aberrant protein production is negligibly low. For these reasons, we consider cHPS2-iPSCs to be an appropriate control for HPS2-iPSCs.We established the HPS2-iPSCs from patient fibroblasts and their gene-corrected cHPS2-iPSCs. Although the mutation in exon 15 persisted in cHPS2-iPSCs, we consider the results obtained from the experiments using cHPS2-iPSCs to be reliable for three reasons. First, the increased CD63 in HPS2-iPSCs was restored in cHPS2-iPSCs, suggesting that AP-3 complex works sufficiently, even in the presence of the exon 15 mutation. Second, HPS2 is an autosomal recessive disease and it was reported that the mother of the donor, who had the nonsense mutation in exon 15 but not in exon 18, had no symptoms of HPS2 . FinallyThe mechanism underlying the formation of giant LBs in HPS is unknown. Since there was a report suggesting that enlarged lysosomes have impaired motility, even in wild-type cells , we inveABCA3 and SFTPB, are typically lethal in neonates because of defective surfactant metabolism  in newborns. In addition, mutations in genes associated with LBs, such as tabolism . In thistabolism . Our fintabolism .+ lung progenitor cells, CPM-based and CD47-based methods were compared  with a 60× objective under 5% COThe use of H9 hESCs was approved by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. The animal experiments were approved by the Animal Research Committee of Kyoto University. The use of human lung samples was approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine.All error bars indicate the SEM. Quantified data represent the findings of three or more independent experiments. The statistical tests used are shown in each legend. All statistical analyses were performed using the Prism7 software program (GraphPad).Y.K. and S.G. conceived and designed the study. Y.K., S.G., S.I., Y.Y., N.S., K.T., S.K., and T.N. performed the experiments. Y.K., S.G., I.A., and A.H. generated HPS2-iPSCs and cHPS2-iPSCs. Y.K., S.G., S.I., Y.Y., T.F.C.-Y., and H.D. contributed to AT2 cell isolation from human lung tissues. Y.K., S.G., S.I., and Y.Y. analyzed the data. Y.K. and S.G. wrote the manuscript through fruitful discussions with and supervision by H.M., I.I., M.H., M.M., and T.H.


Our results lay the foundation for further studies aimed at identifying this protein and determining its role in this particular type of synapse.C-bouton-type cholinergic afferents exert an important function in controlling motoneuron (MN) excitability. During the immunocytochemical analysis of the role of c-Jun in MNs with a monoclonal (clone Y172) antibody against phospho (p)-c-Jun (serine [Ser]63), unexpected labeling was identified in the cell body cytoplasm. As predicted for c-Jun in adult spinal cord, very few, if any MNs exhibited nuclear immunoreactivity with the Y172 antibody; conversely, virtually all MNs displayed strong Y172 immunostaining in cytoplasmic structures scattered throughout the soma and proximal dendrites. The majority of these cytoplasmic Y172-positive profiles was closely associated with VAChT-positive C-boutons, but not with other types of nerve afferents contacting MNs. Ultrastructural analysis revealed that cytoplasmic Y172 immunostaining was selectively located at the subsurface cistern (SSC) of C-boutons and also in the inner areas of the endoplasmic reticulum (ER). We also described changes in cytoplasmic Y172 immunoreactivity in injured and degenerating MNs. Moreover, we noticed that MNs from NRG1 type III-overexpressing transgenic mice, which show abnormally expanded SSCs, exhibited an increase in the density and size of peripherally located Y172-positive profiles. A similar immunocytochemical pattern to that of the Y172 antibody in MNs was found with a polyclonal antibody against p-c-Jun (Ser63) but not with another polyclonal antibody that recognizes c-Jun phosphorylated at a different site. No differential band patterns were found by western blotting with any of the antibodies against c-Jun or p-c-Jun used in our study. In cultured MNs, Y172-positive oval profiles were distributed in the cell body and proximal dendrites. The  In this regard, the execution of complex movements, such as those involved in locomotion, requires the cooperation and refined coordination of different muscles, whose activation is modulated by intricate neuronal circuits in the spinal cord. MNs play a crucial role in these neuromodulatory circuits by adjusting their final motor output and ensuring the accuracy of the movement. MN activity is controlled by excitatory (mainly glutamatergic and cholinergic) and inhibitory (mainly GABAergic and glycinergic) inputs with supraspinal and intraspinal origins  of neurons  or interneurons. Due to the restricted location of cytoplasmic Y172 immunoreactivity in α-MNs and the synaptic-like appearance of Y172-positive profiles located peripherally in cell bodies, we examined their relationship with axon terminals contacting α-MNs and found that Y172 immunoreactive profiles were closely associated with C-boutons. This finding prompted us to analyze in-depth the significance of the unusual immunoreactivity of this antibody against the transcription factor c-Jun found in MNs and the role that its association with C-boutons plays in cholinergic synapse development and maintenance. We examined the compartmentation of Y172 immunoreactivity and its subcellular location in MNs. We analyzed the changes in motoneuronal Y172-positive structures after peripheral nerve injury and in murine models of ALS and SMA. We compared Y172 immunolabeling in the spinal cord with that of other polyclonal antibodies against p-c-Jun. Additionally, we analyzed Y172 immunostaining Generalitat de Catalunya . All experiments were previously evaluated and approved by the Committee for Animal Care and Use of our university. Mice were housed in the University of Lleida Animal Care Facility on a 12-h light/dark cycle with ad libitum access to standard laboratory chow and water.All animal experimentation procedures were performed according to the European Committee Council Directive and the norms established by the G93A (B6SJL-Tg(SOD1-G93A)1Gur/J) mice were purchased from Jackson Laboratory  and maintained as hemizygotes by breeding transgenic males with C57BL/6 females. Smn2B/- mice were obtained by crossing heterozygote male Smn gene knockout mice  with homozygote females with the 2B mutation  20] obtained from fertilized eggs  incubated in the laboratory were also used. The age of the chick embryos was determined by reference to the Hamburger-Hamilton stage series  and xylazine (10 mg/kg), and the sciatic nerve at the femoral level was exposed and transected. The proximal stump of the transected nerve was ligated to prevent spontaneous reinnervation. Postoperative analgesia was achieved by an intraperitoneal (i.p.) injection of buprenorphine (0.05 mg/kg).The spinal cord × neuroblastoma hybrid cell line (NSC-34) was obtained from CELLutions Biosystems Inc. . NSC-34 cells were maintained in a proliferation medium consisting of Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 0.25% penicillin/streptomycin solution  and 2% 200 mM L-glutamine . Cells were subcultured every 2–3 days.For differentiation, cells were seeded in collagen -coated plates. A total of 12,000 NSC-34 cells/well were plated in 4-well culture dishes containing collagen-coated round glass coverslips for immunocytochemical analysis, and 120,000 NSC-34 cells/well were plated in 6-well collagen-coated culture dishes for western blot analysis. Twenty-four hours after seeding, the proliferation medium was exchanged for fresh differentiation medium containing 1:1 DMEM/Ham’s F12 , 1% FBS , 1% modified Eagle’s medium nonessential amino acids  and 1% penicillin/streptomycin . Cells were maintained in the presence or absence of retinoic acid  in differentiation medium, as previously described  molecules were used. Constructs were generated in pSUPER.retro.puro  using specific oligonucleotides  targeting the c-Jun sequence  as follows: shRNA A6, gatccccAAAGGAAGCTGGAGCGGATttcaagagaATCCGCTCCAGCTTCCTTTttttt (forward) and agctaaaaaAAAGGAAGCTGGAGCGGATtctcttgaaATCCGCTCCAGCTTCCTTTggg ; shRNA B4, gatccccAAGTCATGAACCACGTTAAttcaagagaTTAACGTGGTTCATGACTTttttt (forward) and agctaaaaaAAGTCATGAACCACGTTAAtctcttgaaTTAACGTGGTTCATGACTTggg ; and shRNA C2, gatccccATCCGTTTGTCTTCATTTTttcaagagaAAAATGAAGACAAACGGATttttt (forward) and agctaaaaaATCCGTTTGTCTTCATTTTtctcttgaaAAAATGAAGACAAACGGATggg (reverse) targeting the 150-bp region of the c-Jun mRNA sequence.Escherichia coli (E. coli) DH5 cells were transformed with the plasmid. When colonies appeared, they were selected, sequenced to verify that they contained the desired sequence, and purified. Lentiviral constructs were generated by digesting pSUPER-sh with EcoRI and ClaI restriction enzymes to replace the H1 promoter with the H1-short hairpin RNA (shRNA) cassette in the pLVTHM lentiviral plasmid. The pLVTHM vector contained green fluorescent protein (GFP) under the control of an EF-1 alpha promoter for monitoring transduction efficiency. The production of lentiviral particles was carried out as previously described  was added. The medium was changed 24 h later, and the infection efficiency of each experiment was determined by directly counting the GFP-positive cells. The vast majority of cells were GFP positive, with a greater than 90% cell transduction frequency . After 6 days in vitro (DIV), cells were collected and processed for immunocytochemistry and western blot analysis.Adaptors for cloning the oligonucleotides into the BglII/HindII sites of p-SUPER.retro.puro were added as required. 2, and after the coverslip was removed, slides with retained tissue were fixed in methanol at −20°C for 10 min. Serial coronal sections (80-μm thick) of the whole brain and brainstem were also obtained with a vibratome and collected in 0.1 M PB. Cultured cells plated on glass coverslips were fixed with 4% PF in 0.1 M PB, pH 7.4 for 1 h at 4°C. After washing with phosphate-buffered saline (PBS), immunocytochemistry was performed as described below.Spinal cords, brains and brainstems were obtained from anesthetized mice that were transcardially perfused with 4% paraformaldehyde (PF) in 0.1 M phosphate buffer (PB), pH 7.4. Spinal cord samples were postfixed overnight in the same fixative at 4°C and then cryoprotected with 30% sucrose in 0.1 M PB containing 0.02% sodium azide. Serial transverse cryostat sections (16-μm thick) of the lumbar region of the spinal cord were collected on gelatin-coated glass slides and stored at −80°C. In some experiments, transverse spinal cord sections (200-μm thick) were obtained with a vibratome, collected in 0.1 M PB and transferred to a drop of PB placed on Fisherbrand Superfrost Plus microscope slides . A coverslip was then applied to the tissue, which was squashed with forceps. The preparations were then frozen in liquid NFor immunocytochemistry, tissue sections or cultured cells were permeabilized with PBS containing 0.1% Triton X-100 for 30 min, blocked with either 10% normal goat serum or normal horse serum in PBS for 1 h at room temperature, and then incubated overnight at 4°C with an appropriate primary antibody mixture. The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 ; rabbit polyclonal anti-phospho-c-Jun ; rabbit polyclonal anti-phospho-c-Jun ; guinea pig polyclonal anti-synaptophysin 1 ; guinea pig polyclonal anti-vesicular acetylcholine transporter ; guinea pig polyclonal anti-vesicular glutamate transporter 1 ; guinea pig polyclonal anti-vesicular GABA transporter ; mouse monoclonal anti-synaptic vesicle glycoprotein 2A ; mouse monoclonal anti-sigma-1 receptor ; mouse monoclonal anti-Kv2.1 voltage-gated potassium channel ; sheep polyclonal anti-choline acetyltransferase ; rabbit polyclonal anti-ChAT ; rabbit polyclonal anti-neuregulin-1 (NRG1) type III ; mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 ; rabbit polyclonal anti-NRG1 1 α/β 1/2 ; mouse monoclonal anti-Golgi matrix protein of 130 kDa ; mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B ; mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 ; mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 ; and mouse monoclonal anti-calcitonin gene-related peptide .Once washed with PBS, the sections were incubated for 1 h with a combination of appropriate secondary fluorescent antibodies labeled with one of the following fluorochromes (1:500): Alexa Fluor 488, Alexa Fluor 546 , Cy3, or Cy5 . In some experiments, DAPI  was also added to stain the cell nuclei. Finally, the spinal cord sections were counterstained with blue fluorescent NeuroTrace Nissl stain  and mounted using an anti-fading medium containing 0.1 M Tris-HCl buffer (pH 8.5), 20% glycerol, 10% Mowiol, and 0.1% 1,4-diazabicyclooctane. Immunocytochemical controls were achieved by the omission of primary antibodies in parallel-processed sections resulting in no detectable staining.The slides were examined under a FluoView FV-500 or FluoView FV-1,000 Olympus laser scanning confocal microscope . For comparisons, slides from different animals and experimental conditions were processed in parallel for immunocytochemistry and subsequent imaging. The same scanning parameters were used for the acquisition of images corresponding to the different experimental groups. Z-stack confocal images of MN cell bodies  were obtained. When required, Z-stacks are displayed as maximum intensity projections.Brain and brainstem sections that were incubated with the Y172 antibody , were processed for immunoperoxidase staining according to standard ABC procedures . The sections were finally mounted on gelatin-coated slides, air-dried, and coverslipped with DPX. The sections were identified and mapped as described in the Allen Reference Atlas Dong, .Digital images of the entire lumbar region were obtained from every 30th section and analyzed with ImageJ software . Only fluorescent Nissl stained MNs exhibiting a large nucleus, visible nucleolus, and large soma were analyzed. The area and perimeter of MN somata were manually measured. The number, area and fluorescent intensity of Y172-positive profiles, and of profiles expressing the different proteins examined and the degree of spatial association between them, were evaluated in binarized images obtained from multiple immunolabeled sections. Binarized Y172-positive profiles were used as a mask for measuring the immunostaining values of other proteins after splitting multichannel images. For immunofluorescence colocalization analysis, a plugin developed for ImageJ by Pierre Bourdoncle (bourdoncle@ijm.jussieu.fr) was used. In some cases, three-dimensional reconstructions were performed using Bitplane  on a 0.5-μm-thick Z step obtained with a confocal microscope. The digital images were edited using FV10-ASW 3.1 Viewer (Olympus) and Adobe Photoshop CS4 . Although the investigators were not blinded to the experimental condition, to minimize bias, the MN selection for analysis was randomly performed in the fluorescent Nissl channel. In this way, observers were unaware of the pattern of immunostaining to be analyzed in the complementary channels. Experiments were examined by two independent investigators obtaining similar results.Some mice were perfused with either 1% PFA and 1% glutaraldehyde in 0.1 M PB (pH 7.4) for conventional electron microscopy or with 4% PFA and 0.2% glutaraldehyde in 0.1 M PB (pH 7.4) for immunoelectron microscopy.4 and processed for Embed 812 embedding  according to standard procedures. Ultrathin sections were counterstained with uranyl acetate and lead citrate.For conventional electron microscopy, dissected tissues were postfixed in 1% OsOFor ultrastructural Y172 immunolabeling, pre-embedding or post-embedding [freeze-substitution and low temperature embedding in Lowicryl HM20 resin ] procedures were used, as previously described  and then for 1 h with an anti-rabbit biotinylated antibody , visualized using Vectastain Elite ABC (Vector), and incubated in a 0.05% 3,3-diaminobenzidine /0.01% H2O2 mixture. Peroxidase reactive sites were further subjected to silver enhancement to allow visualization by electron microscopy. Briefly, sections were incubated in a solution containing 2.6% hexamethylenetetramine , 0.2% silver nitrate  and 0.2% disodium tetraborate  for 10 min at 60°C. The sections were then rinsed in distilled water and treated with 0.05% gold chloride (Merck) in distilled water for 2 min. Finally, the sections were rinsed, incubated in 2.5% sodium thiosulfate  for 2 min, postfixed with 2% OsO4 in cacodylate buffer for 1 h, and dehydrated in a graded series of acetone. Labeled sections were flat embedded in Embed 812, sectioned and counterstained with uranyl acetate and lead citrate.Briefly, for pre-embedding immunolabeling, vibratome sections were treated with 50 mM glycine in PBS for 30 min, cryoprotected in 30% sucrose in 0.1 M PB and permeabilized with four freeze/thaw cycles in liquid NFor post-embedding, ultrathin cryosections obtained with a Leica EM FC cryo ultramicrotome  were processed according to established procedures  transmission electron microscope.Frozen spinal cords were fragmented and homogenized in blending buffer 1× SR  supplemented with protease inhibitor  and PhosSTOP . The homogenized samples were then heated to 100°C for 5 min and centrifuged at 12,000 rpm for 5 min. The protein concentrations of the supernatants were determined by BIO-RAD Micro DC protein assay . Loading buffer 4× SS  containing 5–10% β-mercaptoethanol  and 15 μg of protein was then loaded onto a 10% or 15% polyacrylamide electrophoresis gel. The proteins were electrotransferred to PVDF  membranes in a Tris-glycine-methanol-buffered solution. The membranes were then blocked with 5% dried skimmed milk in 0.1% Tween 20 and Tris-buffered saline (TBST), pH 8, for 1 h at RT and then extensively washed in TBST. Immunodetection was performed by incubating the membranes overnight at 4°C with the following antibodies: Y172 , rabbit polyclonal anti-p-c-Jun ; rabbit polyclonal anti-p-c-Jun ; rabbit monoclonal anti-c-Jun ; and a mouse monoclonal anti-β-actin, which was used as a loading control. The membranes were washed in TBST, incubated for 60 min at RT with the appropriate peroxidase-conjugated secondary antibodies , washed in TBST, and visualized using the ECL Prime Western Blotting Detection Reagent Detection Kit  following the procedure described by the manufacturer. The quantification of band densities was performed by a using Chemi-Doc MP Imaging System .t-test or either one-way or two-way analysis of variance (ANOVA) followed by post hoc Bonferroni’s test. The level of significance was established at p ≤ 0.05. GraphPad Prism 6 software was used for statistical analysis and the graphical presentation of the data.The data are expressed as the means ± standard error of the mean (SEM). Statistical analyses were performed by student’s 2 (± 40.52 [SEM]) and exhibited strong ChAT immunoreactivity (2) exhibited Y172-negative immunostaining  antibody clone Y172 (hereafter referred to as the Y172 antibody) in combination with an antibody against ChAT  as a MN marker and fluorescent Nissl staining for neuron visualization, was first analyzed in the lumbar spinal cord of adult (P75) CD1 mice by confocal microscopy. Y172 immunolabeling was observed in the form of a bright punctate pattern predominantly distributed in the ventral horn gray matter and particularly in a subpopulation of neurons showing large and angular-shaped somata by Nissl staining . These Yactivity ; all theactivity . In contstaining  and werestaining . In largstaining . Y172 imstaining ; this isstaining , in whicstaining . Image astaining .Due to the striking and unexpected pattern of immunostaining obtained with the Y172 antibody, which had not been previously described for other antibodies against p-c-Jun, we decided to examine the changes in this immunostaining during MN development in CD1 mice . On E16 We next explored whether a similar pattern of immunoreactivity can be observed with the same antibody in other animal species, such as rats and chickens. In both species, the Y172 antibody exhibited a comparable pattern of immunostaining and distribution in MNs and spinal cord gray matter as that seen in adult mice .n = 31 MNs) Y172-positive profiles peripherally located in MN somata were in close contact with synaptophysin-positive puncta. We then assessed whether these Y172-positive profiles were associated with a specific type of afferent inputs by immunocytochemistry by combining the monoclonal antibody with anti-VAChT, -VGluT1 and -VGAT antibodies . Pixel profile analysis indicated an overlap between S1R and Kv2.1 signals and Y172 immunostaining  and displayed a reticular micropunctate pattern compared to the more compact and homogeneous appearance of Y172 puncta  and VAChT . We founstaining , indicatstaining . As the 2 puncta .We also explored whether specific labeling in spinal cord MNs with the Y172 antibody is also present in cranial MNs. We found that MNs from facial (CN VII), ambiguous (CNX), hypoglossus (CN XII), and trigeminal (CNV) nuclei displayed an identical punctate cytoplasmic pattern of Y172 immunoreactivity. This appeared to sometimes be associated with a weak nuclear Y172 signal, as expected for p-c-Jun. In contrast, MNs from nuclei that innervate extrinsic ocular muscles, such as the oculomotor (CN III), trochlear (CNIV), and abducens (CNVI) nuclei, exhibited higher nuclear Y172 positivity, but the punctate cytoplasmic pattern was virtually absent. This is consistent with the described absence of C-bouton afferents in ocular MNs .Peripheral nerve interruption results in noticeable structural and metabolic changes in axotomized MNs  and SMA . Again, at these time points, SOD1G93A MNs that were degenerating and showed intense Y172 immunoreactivity in their nuclei were those that exhibited scarce immunoreactive cytoplasmic profiles; these MNs alternated with MNs that displayed apparently healthy morphology and profuse cytoplasmic Y172 immunolabeling. As expected from our previous analysis  compared to that in age-matched WT MNs  was hardly visible when confocal images were captured using the same scanning parameters as those utilized for the Y172 antibody. However, when the microscopy settings were modified to attain a higher sensitivity for detection, the polyclonal antibody exhibited an immunocytochemical pattern similar to that obtained with the Y172 antibody . As expeWe next examined whether the Y172 antibody is able to detect proteins other than p-c-Jun (Ser63) in spinal cord extracts by western blot analysis. We compared the protein band pattern detected by the Y172 antibody with that identified with the other abovementioned antibodies against p-c-Jun and, also, against c-Jun . The Y17in vitro models such as NSC-34 MN-like cells and cultured CD1 MNs. Due to the highly restrictive immunostaining found with the Y172 antibody in MNs in vivo, a similar pattern of immunolabeling in vitro could provide us enough protein for purification and further analyses.We next analyzed whether Y172 immunoreactivity could also be observed in in vivo, NSC-34 cell differentiation resulted in a progressive decrease in nuclear Y172 immunolabeling, which was accompanied by a gradual emergence of positive cytoplasmic spots. To explore whether Y172-immunoreactive spots in differentiated NSC-34 cells display a spatial association with VAChT-positive puncta, double immunofluorescence analysis with Y172 and anti-VAChT antibodies were performed. After 2 days of differentiation, VAChT-immunostaining was already present in NSC-34 cells in the form of a weakly positive diffuse staining distributed in the cytoplasm; no clear and defined concentrations of VAChT-positive puncta were observed in the periphery of cell bodies or proximal dendrites. This pattern of VAChT immunostaining remained unchanged after 4 and 8 days of cell differentiation. This finding is consistent with previous reports  examined.Differentiated NSC-34 cells resemble mature MNs based on several morphological and physiological features  targeting a specific site of the c-Jun mouse sequence and transduced purified cultured MNs. The c-Jun shRNA knockdown efficiency was determined by western blot analysis with the Y172 antibody , a new identified protein specifically located in retina ribbon synapse, is encoded by the same gene as CtBP2, the CtBP2/RIBEYE gene, when an alternative promoter is used. CtBP2 is a transcriptional repressor ubiquitously expressed, whereas RIBEYE is expressed in a tissue-specific fashion and is involved in synaptic vesicle transport and exocytosis  displayed C-bouton labeling. However, in western blot analyses of spinal cord extracts, we did not find significant differences between p-c-Jun (Ser63) and (Ser73) antibodies. Therefore, we conclude that the synaptic-specific protein detected by the Y172 antibody was not identified by western blot, probably due to its scarce representation in the whole spinal cord extracts used for the analysis. In fact, MNs represent less than 10% of the neuronal population of the spinal cord (Arce et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Committee for Animal Care and Use of University of Lleida.JC and JE conceived and designed the research. AG, PC, OT, AC, SH, SS, LP, RS, JC, and JE performed the research. AG, PC, OT, JC, and JE analyzed the data. JC, JE, OT, and AG wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XN declared a past co-authorship with several of the authors OT, AC, LP, JE, JC to the handling Editor.


The genetic variant rs2237895, located in the Potassium Voltage-Gated Channel Subfamily Q Member 1 (KCNQ1) gene, has been replicated to be associated with type 2 diabetes mellitus (T2DM) susceptibility, but the relationship with lipids is conflicting. Furthermore, the common genetic predisposition to T2DM and lipids was not fully detected.  In total, 5839 individuals (2220 were T2DM patients) across 2885 families were included. The effect of rs2237895 on T2DM and lipids was estimated using linear regression and logistic regression models after adjustment for multiple covariates. Mediation analysis was then used to test whether KCNQ1 participated in T2DM pathogenesis via lipid-mediated pathways. P < 0.001) increased T2DM risk. Moreover, it was correlated with 5% , 4% , 2% , and 2%  higher total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A, and apolipoprotein B (Apo-B) concentrations, respectively. Nevertheless, the genetic susceptibility for higher T2DM risk was correlated with higher high-density lipoprotein cholesterol (HDL-C) level . Mediation analysis showed only TC, LDL-C, and Apo-B had small significant mediated effects, with 2.9%, 2.3%, and 3.1% of the total effects of rs2237895 on T2DM being mediated by them, respectively.  Per allele-C of rs2237895 was associated with 17% (11-23%,  KCNQ1 had pleiotropic effects on lipids and T2DM, and the unexpected genetic effect on association of HDL-C with T2DM was observed, indicating the different pathways to lipids and T2DM. Further research studies are needed to verify potential biological mechanisms. Type 2 diabetes mellitus (T2DM) continues to be an epidemic global health issue across the world. An estimated 425 million adults worldwide had diabetes in 2017, and that figure is expected to reach 642 million by 2040 .+ channel subunit KvLQT1, is involved in the repolarization of the cardiac action potential as well as water and salt homeostasis [Genetic factors play an important role in the pathogenesis of T2DM , 5. So feostasis  and insueostasis . Rs22378eostasis –12.Previous studies have explored the relationships between KCNQ1 SNPs and lipid profiles, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), but with conflicting results. It has been demonstrated that KCNQ1 gene was associated with lipid parameters, including TG, HDL-C, and apolipoprotein A (Apo-A) among middle-aged Chinese Han population . AnotherLipid metabolism is tightly intertwined with glucose metabolism, insulin secretion, and T2DM , 19. DysTherefore, the aim of the present study was to explore the associations between rs2237895 and lipid profiles ) and T2DM in Chinese to interpret the pleiotropic genetic effects of KCNQ1.The study population was part of the Fangshan/Family-based Ischemic Stroke Study in China (FISSIC) program from 2005 to 2017 in Fangshan district, in the southwest of downtown Beijing, China. The relevant details of the FISSIC program have been described before . Until t2 (kg/m2)).Fasting for at least 8 hours, fasting venous blood was drawn for biochemical collection and measurements. Six common lipid profiles were TC, TG, LDL-C, HDL-C, Apo-A, and Apo-B. The status of T2DM was defined if at least one of the criteria was met: (i) fasting plasma glucose (FPG) ≥ 126 mg/dL, (ii) hemoglobin A1c (HbA1c) > 6.5%, or (iii) ongoing antidiabetic medication therapy. Other information was obtained by structured questionnaires and physical examinations. The questionnaires were completed by face-to-face interviews, including the basic characteristics , smoking and drinking status , medical history , and medication use history. The height and weight were measured by the physical examinations to calculate BMI (weight/heightAmplification of the genomic DNA fragment was performed using an Amplicon ThermoEx 500 PCR instrument followed by a mass spectrometry system using a matrix-supported laser release/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for high-throughput, fast, and accurate genotypic analysis. Negative (blank) and positive controls  were provided. To validate genotyping procedures, the reproducibility of the 5% random sample reached 100%, and the call rate was over 95%.t-test was adopted to compare continuous variables. Categorical variables were described as proportions, and McNemar's chi-squared test was used to compare groups. Hardy-Weinberg equilibrium (HWE) was tested using chi-squared statistic. HWE P value for rs2237895 was 0.156, showing no violation for rs2237895. Then, an additive genetic model was assumed for rs2237895 where the genotype value was coded as the count of risk allele-C. The additive model showed a linear relationship between the number of risk alleles and disease risk.Continuous variables were described as the mean ± standard deviation, and the paired To estimate the association of rs2237895 with the lipid parameters, we applied a general linear regression model. Associations between genotype and T2DM (dichotomous outcomes) were tested using a logistic regression model. All models were adjusted for age, sex, smoking and drinking status, BMI, hypertension, and coronary heart disease. The results were expressed as the percent change (PC) and 95% confidence intervals (CIs) in per allele-C of rs2237895 associated with increase in lipid concentrations and T2DM risk or each 1 mmol/L lipid concentrations associated with increase in T2DM risk. Subgroup analyses were conducted separately in subjects with BMI ≥ 24 and BMI < 24, smokers and nonsmokers, and drinkers and nondrinkers.d∗(ORd − 1)/(ORd∗ORi − 1), where ORd is the direct effect odds ratio and ORi is the indirect effect odds ratio [P value < 0.05 was considered significant. All analyses were performed using R Programming Language .Counterfactual-based mediation analysis was then used to test whether KCNQ1 participated in the pathogenesis of T2DM via lipid-mediated pathways. The mediation contains two regressions. The first for the binary outcome (T2DM) was regressed on the exposure (rs2237895), the proposed mediator (lipid parameters), and other relevant covariates. The other regression was for the mediator, regressing on the exposure and the same covariates. Both regressions could obtain the effects mediated by lipid (indirect effect) and by pathways other than those involved in mediators (direct effect). The proportion mediated was equal to ORds ratio . All staThe baseline characteristics of study population were summarized according to the T2DM patients and control groups . IndividP = 0.003), 14% , 7% , and 27%  increased risk of T2DM, respectively. In contrast, each 1 mmol/L increase in HDL-C conferred a 25%  decrease in T2DM risk , 4% , 2% , 2% , and 2%  higher levels of TC, LDL-C, HDL-C, Apo-A, and Apo-B, respectively. Under an additive model, per allele-C was significantly associated with 17%  increased T2DM risk after adjustment for the covariates. When adding the lipid parameters as covariates, a positive correlation was still found between rs2237895 and T2DM .The relations between rs2237895 and different lipid parameters and T2DM are presented in P = 0.011) increased risk of T2DM in BMI ≥ 24. Per allele-C of rs2237895 was associated with 23%  increased risk of T2DM in smokers compared with 14%  increase in nonsmokers. In drinkers, per allele-C of rs2237895 was associated with 20%  increased risk of T2DM, compared to 11%  increase in nondrinkers after adjusting for conventional risk factors  . Women'sIn the subgroup analysis, the associations between rs2237895 and some lipid parameters disappeared, which might result from insufficient sample size and inadequate power . HoweverThe main strength of the study was from the family-based design, which offered similar environmental exposure, thus attenuated population stratification . AlthougIn conclusion, KCNQ1 SNP rs2237895 had pleiotropic effects on lipids  as well as T2DM risk. It was remarkable that we observed paradoxical genetic association of HDL-C with T2DM, where the genetic susceptibility for higher T2DM risk was correlated with higher HDL-C level. Our findings added important evidence for the genetic predisposition to both lipids and T2DM.


Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-secreting islet β cells by autoreactive T cells. Non-obese diabetic (NOD) mice are the widely used animal model for human T1D. Autoimmunity in NOD mice is associated with particular major histocompatibility complex (MHC) loci and impaired islet autoantigen expression and/or presentation in the thymus, which results in defects in both central and peripheral tolerance. It has been reported that induction of mixed chimerism with MHC-mismatched, but not MHC-matched donor bone marrow (BM) transplants prevents the development T1D in NOD mice. We have reported that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to generate thymic epithelial progenitors (TEPs) that further develop into thymic epithelial cells (TECs) in vivo to support T cell development.g7 B6 mice. The mice were monitored for T1D development. The pancreas, spleen, BM, and thymus were then harvested from the mice for evaluation of T1D, insulitis, chimerism levels, and T cells.To determine whether transplantation of MHC-mismatched mESC-TEPs could prevent the development of insulitis and T1D, NOD mice were conditioned and injected with MHC-mismatched B6 mESC-TEPs and MHC-matched BM from H-2Transplantation of MHC-mismatched mESC-TEPs and MHC-matched donor BM prevented insulitis and T1D development in NOD mice. This was associated with higher expression of proinsulin 2, a key islet autoantigen in the mESC-TECs, and an increased number of regulatory T cells.Our results suggest that embryonic stem cell-derived TEPs may offer a new approach to control T1D.The online version of this article (10.1186/s13287-019-1347-1) contains supplementary material, which is available to authorized users. Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-secreting islet β cells by autoreactive T cells . Non-obeTransgenic expression of diabetes-protective MHC alleles in the thymus has been reported to prevent T1D development in NOD mice , 14. HowAnother strong T1D genetic risk factor is associated with a variable number of tandem repeat minisatellites, upstream of the insulin gene that modulates thymic (but not pancreatic) expression of this antigen –25. In hT cell development occurs in the thymus, and is critically dependent on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component. However, TECs undergo both qualitative and quantitative loss over time, which is the major factor for age-dependent thymic involution. We have reported that mouse embryonic stem cells (ESCs) (mESCs) can be selectively induced to generate thymic epithelial progenitors (TEPs) in vitro . When trIn this study, we determined the ability of mESC-TEPs to prevent T1D. We show here that transplantation of MHC-mismatched C57BL/6 (B6) mESC-TEPs and MHC-matched nonautoimmune donor BM prevents insulitis and T1D development in NOD mice that were pre-conditioned with anti-CD3/CD8 antibodies (Abs). This is associated with establishing diabetes-protective MHC alleles, increasing the expression of proinsulin 2 (Ins2) in the thymus, and improving the thymic microenvironment, thereby the restoration of defective negative selection to insulin in NOD mice. In addition, transplantation of the mESC-TEPs leads to an increased number and function of Tregs in the thymus and the spleen.g7 B6 mice were purchased from The Jackson Laboratory . All experimental procedures involving mice were approved by the University of Connecticut Animal Care and Use Committee and were conducted in accordance with NIH guidelines.Female NOD/LtJ, B6, and H-2+ B6 mESC lines  were first differentiated into definitive endoderm (DE), and then TEPs as described  thymidine (1 μCi/well)  12 h before harvest. Incorporation of [3H] thymidine was measured by liquid scintillation spectroscopy . The suppression index = [proliferation  without Tregs − CPM with Tregs]/CPM without Tregs.Effector T cells (Teffs)  was determined to be significant.+ T cells induced stable permanent mixed chimerism without GVHD [d, I-Ag7, CD45.1) were conditioned with anti-CD3 and anti-CD8 Abs on days − 8 and − 3. On day 0, the mice were injected i.v. with BM and CD4+ T cell-depleted spleen cells (20 × 106 each) from MHC-mismatched B6  or MHC-matched congenic H-2g7 B6  mice. The control mice were given a conditioning regimen only.It has been reported that BM cells in combination with donor CD8out GVHD . Dr. Zenout GVHD . We haveThe mice were then monitored for T1D development by analyzing blood glucose levels. On day 100, the mice were euthanized and the blood, pancreas, spleen, BM, and thymus of the mice were harvested for evaluation of T1D, insulitis, and chimerism levels. As shown in Fig. + T cells and B220+ B cells . There were no significant differences in the chimerism levels between the MHC-matched and MHC-mismatched BMT mice. In both groups, the spleens contained 30–40% host-type TCRTaken together, our results confirmed the previous reports that mixed chimerism was present in the NOD mice conditioned with anti-CD3/CD8 Abs and followed by either MHC-matched or MHC-mismatched BMT. Induction of mixed chimerism with MHC-mismatched but not matched BM transplants prevents insulitis and T1D development in these mice.+ T cell-depleted spleen cells (20 × 106 each) from MHC-matched H-2g7 B6 mice as described above. Groups of the BMT recipient were also injected i.t. with MHC-mismatched B6 mESC-TEPs (EpCAM1+ cells) or mESC-control cells (EpCAM1− cells).We then determined whether transplantation of MHC-mismatched mESC-TEPs could prevent the development of insulitis and T1D in NOD mice. To this end, NOD mice were conditioned with anti-CD3 and anti-CD8 Abs and injected i.v. with BM and CD4− cells) did not reduce the incidence of T1D. In contrast, transplantation of mESC-TEPs prevented the development of T1D with none of the mice having T1D. Furthermore, 78% and 73% of the residual islets in the MHC-matched BMT control mice or mESC-control cell-transplanted mice had insulitis, respectively  and mTECs (CD45−EpCAM+Ly51−) in vivo [−EpCAM+) by qRT-PCR. By using GFP+ B6 mESCs to separate mESC-TECs (GFP+ CD45− EpCAM+) and host TECs (GFP− CD45− EpCAM+), we found that the expression level of Ins2 in mESC-TECs was about twofold higher than that in host TECs (Fig. Many studies have shown that insulin/proinsulin is the key autoantigen in initiating the autoimmunity of T1D –25. Proi in vivo . We analIt has been reported that, in MHC-mismatched BM-transplanted NOD mice, the residual host-type peripheral T cells were anergic caused by the upregulation of PD-1 and downregulation of IL-7Rα . We deteIn this study, we demonstrate that induction of mixed chimerism with MHC-matched BM transplants from nonautoimmune donors cannot prevent the development of insulitis and T1D in NOD mice, consistent with other reports , 21. In It has been reported that NOD or T1D patients have a defect in central tolerance –12, 41. g7 [g7 has a weak and unstable peptide-binding property, leading to the defect in thymic negative selection [g7 prevent T1D development [The autoimmunity in NOD mice is related to the I-Ag7 . It has election , 10. Conelopment , 14. TheWe have also shown that mESC-derived TECs expressed a higher level of Ins2 than host TECs in the NOD mice. Although multiple inlet autoantigens have been implicated in T1D, insulin/proinsulin is a major autoantigen associated with T1D –25. It hIn addition to the deletion of autoreactive T cells in the thymus, peripheral tolerance also plays a role in the prevention of insulitis and T1D. Our data suggest that the mechanisms of the peripheral tolerance induced by the two approaches seem also to be different. In the MHC-mismatched BMT mice, the residual host-type peripheral T cells were anergic due to upregulation of PD-1 and downregulation of IL-7Rα. In contrast, transplantation of mESC-TEPs did not lead to the upregulation of PD-1 and downregulation of IL-7Rα. Instead, it resulted in an enhanced Treg development in the thymus, leading to an increased number of Tregs in the periphery.Reduced frequency and/or function of Tregs in both NOD mice and T1D patients were observed by some groups , 39, altIn this study, we have used mESCs to show the feasibility of ESC-TEPs/TECs to ameliorate insulitis and T1D in NOD mice. Using mESC-derived cells also facilitates mechanistic studies. In the future, we will also test the ability of human ESC- and iPSC-derived TEPs/TECs to prevent and treat T1D. Our present studies using NOD mice have some limitations. For example, we transplanted mESC-TEPs into mice prior to the onset of insulitis. Therefore, this protocol was designed to prevent T1D. In future studies, we will determine the ability of ESC- and iPSC-TEPs/TECs to reverse or treat T1D by transplantation of the cells after the onset of insulitis.In summary, we have demonstrated that transplantation of MHC-mismatched mESC-TEPs prevents insulitis and T1D development in NOD mice. This is likely due to the restoration of defects in both central and peripheral tolerance in NOD mice. Our findings justify further evaluation of human ESC- and iPSC-derived TEPs/TECs in the prevention and treatment of T1D in both animals and human patients.Additional file 1:Figure S1. Isotype antibody staining for Figs.


Recent studies have revealed that aberrant m6A modifications are involved in many diseases.Large-scale genome sequencing projects have identified many genetic variants for diverse diseases. A major goal of these projects is to characterize these genetic variants to provide insight into their function and roles in diseases. N6-methyladenosine  are accepted as the input data, and the output includes an interactive table that contains the genetic variants annotated by m6A function. In addition, statistical diagrams and a genome browser are provided to visualize the characteristics and to annotate the genetic variants.In this study, we present a user-friendly web server called “m6ASNP” that is dedicated to the identification of genetic variants that target mWe believe that m6ASNP is a very convenient tool that can be used to boost further functional studies investigating genetic variants. The web server “m6ASNP” is implemented in JAVA and PHP and is freely available at [60]. Due to rapid improvements in high-throughput sequencing technology, the cost and time requirements of these technologies have been greatly reduced, which has triggered the explosive growth of high-throughput sequencing data associated with various diseases. The major goal of these high-throughput sequencing studies is to identify disease-causing variants. However, distinguishing the few disease-causing variants from the majority of passenger variants remains a major challenge. Computational methods that accurately interpret and prioritize the large amount of variants are urgently needed.Many types of variants have different effects on the function of genes. Nonsynonymous variants, which alter the amino acids in a protein sequence, are among the most studied classes of variants. Alterations in the protein sequence can cause protein dysfunction due to a variety of different mechanisms. For example, variants in critical sites of the catalytic domain may affect protein catalytic functions  and variCompared to nonsynonymous variants, synonymous variants are neglected by most studies investigating diseases, particularly studies investigating tumors . These v6A) modification is among the most highly abundant in post-transcriptional modification [6A modification is closely related to multiple diseases. Recently, FTO, an m6A demethylase, has been found to play an important role in the development of recessive lethality syndrome [6A regulation can lead to individual developmental retardation [6A modification was closely related to cancer development. It was shown that abnormal modification of m6A and its regulators can lead to leukemia [6A modification, providing new perspective for understanding the variants, particularly for synonymous variants, thus helping to find more disease-causing variants.The post-transcriptional modification of mRNA is also an important post-transcriptional regulatory mechanism, and N6-methyladenosine  within the exonic regions were preserved for subsequent analysis. A total of 13,079,416 and 2,668,046 SNVs were collected in human and mouse, respectively. To investigate the potential role of these SNVs in reshaping the m studies , two PA-eriments , and 2446A-associated variants in post-transcriptome regulation, the RNA-binding protein (RBP) binding sites from starBase2 [In order to identify the potential roles of mtarBase2  and CLIPtarBase2 , the miRtarBase2 , JohnsontarBase2 , dbGAP [tarBase2 , GAD [49tarBase2 ) to perf6A sites. Nucleotides in the DRACH motif around the N6-methyladenosine were dominated for classification  molecular functions  or a simplified tab delimited file are supported as input data Fig. . As an eble Fig. . Of the ons Fig. . The seqces Fig. . The “GGnts Fig. .6A-associated variants. Among these m6A-associated variants, 6,235 located at or near the m6A sites from miCLIP experiments and 55,381 located at or near the m6A sites from MeRIP-Seq experiments. To characterize m6A-associated variants predicted by m6ASNP, we performed a systematic comparison between m6A-associated variants and non-m6A-associated variants (non-m6A variants). We found that m6A-associated variants were enriched in protein-coding genes  and significantly concentrated in CDS and 3′UTR . To further explain the functional role of m6A-associated variants, we divided the predicted m6A-associated variants into two groups: the functional gain and functional loss variants. The conservation analysis was performed on these two groups, and the results were compared to non-m6A variants in both CDS and UTR regions , Familial breast cancer  and hereditary nonpolyposis colorectal cancer  were the top enriched disease types (Genome=wide association studies (GWAS) have revealed many disease-related variants. However, the pathogenesis mechanisms for most of these disease-related variants had not been known. We found 1,919 mse types . Our fin6A-associated variants predicted by m6ASNP, 59.86% and 25.67% are synonymous variants in mouse dbSNP and human dbSNP, respectively. By using m6ASNP, we identified many m6A-associated synonymous variants that have been shown to be disease related. For instance, rs139362268, a synonymous variant of PALB2, is related to breast cancer and pancreatic cancer. Interestingly, we observed that rs139362268 occurred in the m6A site of PALB2, in which m6A peaks were detected in six MeRIP-Seq experiments  with a bracket or dot. For example, a given m6A nucleotide with the sequence TTCCGGGACTGGCAGG could be represented as ((()))((.)). Next, we extracted the secondary structure triplet, formed by the structure state of the three adjacent nucleotides obtained from the predicted RNA structure. The number of occurrences of each triplet in the sequence was counted and normalized to produce a 27-dimension feature vector. Combining all the primary sequences and secondary structure features, we constructed an 87-dimension vector for each m6A residue. These vectors were subsequently used as the input for a random forest classifier for training and prediction.The sequences of the flanking regions 30 nucleotides upstream and downstream of a given m studies , 58, spe studies , we firsThe random forest classifier for human and mouse were trained separately on the above-collected training set. The tree number was optimized as 500 and the features used for each splitting were set to 9. To assess the performance, we used 4-, 6-, 8-, and 10-fold cross-validation on the training set. The additional test set was also applied in our study to evaluate the robustness. The sensitivity, specificity, and Matthew's correlation coefficient were used to measure the predictor's performance.6A site prediction model, we then developed a computational pipeline to predict the effect of variants on m6A modification. First, variants were mapped to known transcripts. The wild-type and mutant form of the transcript sequences were then generated for m6A site prediction. For an m6A site that occurred in the wild-type transcript and disrupted in the mutant transcript, we defined it as an m6A-associated loss variant. The m6A-associated gain variant was conversely formed. To measure the altered degree of m6A modifications, equation 6A alterations between reference and mutant samples and RF_Score is the predicted score of a given m6A site from the random forest model. Obviously, alteration scores higher than 0 represent m6A-gain alterations, while scores lower than 0 represent m6A-loss alterations. In some m6A-associated loss variants, alteration scores were assigned to MAX, which means that the core AC motif is destroyed by genetic variants, leading to complete losses of m6A at those sites.Based on the m6A modification. m6ASNP was implemented using JAVA and PHP and is freely accessible at [To provide convenience to the research community, we developed a web server called “m6ASNP” to specifically predict the effect of variants on msible at .6A-seq, and MeRIP-seq data, we then combined them with the SNV data from dbSNP and performed m6A-association prediction using m6ASNP. Following the same procedure proposed in our previously published work [6A-associated variants for subsequent analysis.Based on miCLIP-seq, PA-mhed work , we cons6A-associated variants derived from miCLIP-seq and PA-m6A-seq experiments. Notably, the PA-m6A-seq can only detect m6A signal in a resolution of ∼23 nt. Therefore, in order to obtain precise modification sites, we scanned through all the peak regions and extracted adenosine sites that conformed to DRACH motif as the final m6A sites. On this basis, we retained the variants that located near the m6A sites as the m6A-associated variants.The first annotation was the high-confidence-level data that contained the m6A peaks in each study separately. In general, in MeRIP-seq experiments, if a given region is identified as enriched in most of the adopted methods, it is more likely to be a true modification signal. Therefore, to obtain reliable m6A peaks, a tool called MSPC [6A-associated variants that significantly change the DRACH motif.The second annotation was the medium-confidence-level data. We first downloaded all the published MeRIP-seq data from the GEO database. According to the standard analysis pipeline for MeRIP-seq data, we applied MACS2 , MeTPeakled MSPC  was then6A-associated variants from all collected genetic variants.The third annotation was the low-confidence-level data, where we used the whole transcriptome sequences for prediction. With a high threshold, m6ASNP will predict the potential m6A-associated variants for human. Another 935 high-confidence-level, 9,404 medium-confidence-level,and 17,739 low-confidence-level data were also constructed for mouse.In summary, we constructed 13,703 high-confidence-level, 54,222 medium-confidence-level, and 243,880 low-confidence-level m6A-associated variants were annotated by the transcript structure, including the CDS, 3′ UTR, 5′ UTR, start codon, and stop codon. For the annotation of noncoding RNA DASHR [RRID:SCR_003152) [6A-associated variants were more preferentially distributed in specific transcript structures, we calculated the proportion of variants that located in a given transcript structure. In order to avoid bias, only the variants that were annotated in mRNA were used, and the proportion in 5′-UTR, CDS, and 3′-UTR were calculated. A 2-tailed proportion test was then adopted to compare the proportion difference between m6A-associated variants and non-m6A variants. In addition, in order to evaluate their conservation scores and deleteriousness, we further annotated the m6A-associated variants by ANNOVAR (updated 1 February 2016)  [6A-associated variants and non-m6A variants in the same exon. As the selective pressures were quite different in protein-coding sequences and untranslated regions, the above comparison was carried out separately for the CDS and UTR regions. Specifically, the conservation scores were calculated by phastCons with 100-way and 60-way gene conservation profiles for the human and mouse, respectively [All the identified mNA DASHR , miRBase_003152) , GtRNAdb_003152) , and piR_003152)  were use_012821) . To avoiectively . The delectively , PolyPheectively , and FATectively ). We defRRID:SCR_003076) to obtain the LD mutations using a parameter r2 > 0.8 in at least one of the four populations from CHB, CEU, JPT, and TSI. Then, we selected all m6A-associated variants by mapping the variants to GWAS disease-associated SNPs and their LD mutations. Moreover, we collected ClinVar data to annotate the m6A-associated variants with specific functions.A linkage disequilibrium (LD) analysis was performed for each GWAS disease-associated SNP. We used Haploview . Second, because certain m6A-associated variants randomly occur within the YTHDF2-binding regions, we estimated the background count of m6A-associated variants for YTHDF2 (defined as BN). Thus, we extracted the longest transcript for each gene from the gene annotation files. The weight of the ith gene was defined as follows:n was the total number of genes annotated and L(i) was the length (bp) of the ith gene. Then, we extracted the same-length reads of all YTHDF2-binding regions, which was defined as BN, using weighted random sampling of all transcripts collected above. We repeated this procedure 50,000 times and then obtained the frequency RBPF when NB was greater than RBPN in the cycle. This frequency may be regarded as an estimation of the probability that observing BN greater then RBPNin random condition. Next, the Benjamini-Hochberg method was applied to control the false positives. An adjusted RBPF less than 0.05 was considered a small probability event, suggesting that the m6A-associated variants were more likely to occur in the RBP-binding regions of YTHDF2. All significant RBPs are listed in First, we calculated the number of mProject name: m6ASNPhttps://m6asnp.renlab.orgProject home page: https://github.com/RenLabBioinformatics/m6ASNPRRID:SCR_016048Operating system(s): platform independentPrograming language: PHP, java, javascriptLicense: GPLv3et al. and Ke et al. are available in the supplementary data. These and snapshots of the code are also available in the GigaScience GigaDB repository [The training data and test data collected from Linder pository .Supplementary Figure S1: The feature contribution of the human and mouse model. Distribution plot of the feature's Gini importance for both (A) human and (B) mouse model. The prediction capabilities of different combination of features for (C) human and (D) mouse model.Supplementary Figure S2: A systematic comparison of the m6A-associated variants and non-m6A variants. (A) Proportional distribution of the variants at different m6A confidence levels and non-m6A variants located in the CDS and 3′ UTR. A two-tailed test of population proportion was performed to assess significance. (B) Boxplots show the gene isoforms of the m6A-associated variants and non-m6A variants in different databases. One-sided Wilcoxon signed-rank test was performed to determine the significance. “**” indicates a significance level of P ≤ 0.01, while “*” indicates P ≤ 0.05.Supplementary Figure S3: The characteristics of m6A-associated variants predicted by m6ASNP. (A) The conservation differences between functional gain and functional loss variants. (B) The comparison of mutation deleteriousness between functional gain and functional loss variants.Supplementary Figure S4: Association analysis of m6A-associated variants. (A) An example of m6A-associated variants in disease. The red rectangle in exon 10 represents a synonymous mutation in PALB2, i.e., rs139362268, while the green rectangle represents the m6A site. The 1 to 6 numbering indicates the different samples, followed by HepG2, GM12878, Momo-mac-6, HeLa, shMETTL14 in A549 and shGFP in A549. MeRIP-seq peak tracks of input, and the IP samples were scaled to the same level and colored in red and blue. (B-C) Proportional distribution of different levels of m6A-associated variants and non-m6A variants located within the RBP-binding regions and miRNA target regions. A two-tailed test of population proportion was performed to assess significance. “**” indicates a significance level of P ≤ 0.01, while “*” indicates a significance level of P ≤ 0.05.Supplementary Table S1: The distribution characteristics of m6A-associated variants in different transcript structures.Supplementary Table S2: Significant disease phenotypes in m6A-associated.Supplementary Table S3: Significant RBPs in m6A-associated variants Supplementary Table S3. Significant RBPs in m6A-associated variants.Supplementary Table S4: Significant miRNAs in m6A-associated variants.Supplementary Data 1. Single nucleotide resolution m6A sites .6A, N6-methyladenosine; RBP, RNA-binding protein; SNP, single nucleotide polymorphism; VCF, Variant call format.GO, Gene ontology; GWAS, Genome-wide association study; LD, Linkage disequilibrium; mNot applicable.The authors declare that they have no competing interests.GIGA-D-17-00348_Original_Submission.pdfClick here for additional data file.GIGA-D-17-00348_Revision_1.pdfClick here for additional data file.GIGA-D-17-00348_Revision_2.pdfClick here for additional data file.GIGA-D-17-00348_Revision_3.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Response_to_Reviewer_Comments_Revision_1.pdfClick here for additional data file.Response_to_Reviewer_Comments_Revision_2.pdfClick here for additional data file.Reviewer_1_Report_ -- Yun-Gui Yang1/3/2018 ReviewedClick here for additional data file.Reviewer_1_Report_(Revision_1) -- Yun-Gui Yang2/11/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Bastian Linder18 Jan 2018 ReviewedClick here for additional data file.Reviewer_2_Report_(Revision_1) -- Bastian Linder2/22/2018 ReviewedClick here for additional data file.Supplemental materialClick here for additional data file.