CFTR) gene mutation analysis has been implemented for Cystic Fibrosis (CF) carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD). Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM) curve analysis, allele-specific PCR (AS-PCR) and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.Cystic fibrosis transmembrane conductance regulator ( CFTR) gene . CFTR mutations are also found in infertile males with congenital bilateral absence of the vas deferens (CBAVD). About 80% of men with CBAVD have at least one CFTR mutation (CFTR gene has become part of the routine for diagnosis of CF and/or CBAVD.Cystic fibrosis (CF) is one of the most common autosomal recessive disorders among northern European descendants. It affects lung, pancreas, liver, intestine and male reproductive systems. Diagnosis of CF is based on clinical manifestations plus sweat chloride tests, transepithelial nasal potential difference (NPD) measurements, or mutation analysis in cystic fibrosis transmembrane conductance regulator (TR) gene . Moleculmutation . TherefoCFTR testing  constitutes a disease-causing CF mutation (cis-configuration) or different parent , if 5T is detected in R117H positive samples. Because R117H and poly-T alleles are about 18 kb apart, long-range PCR has also been developed to determine the cis- or trans-relationship of R117H and 5T alleles to circumvent testing unaffected parents  technology, laboratories started to develop their own NGSCFTR testing. Alignment of the sequence reads in the poly-T region is extremely challenging as there are three common poly-T alleles and variable numbers of TG repeats at the 5′ of the poly-T tract in each individual allele. Commonly available NGS commercial software cannot align the region properly. End users have to write their own script in order to have the correct alignment in the poly-T region  is not recommended for routine carrier screening. For this reason, some commercial assays mask the results of the poly-T tract and only provide poly-T results when the samples are R117H positive. Others have the poly-T assay in a separate reaction from the rest of the 23 mutations to serve as a standalone reflex test. When a clinician wants to test the poly-T tract for a male patient with clinical indication of CBAVD, a standalone poly-T test  would be ordered, because it is impossible to obtain the poly-T tract results with R117H negative samples if laboratories only run commercial T region . This woThere are standalone poly-T assays developed for clinical research or testing, such as heteroduplex , High ReIn contrast, interpretation of AS-PCR results is simpler. Presence or absence of the AS-PCR products would indicate the specific variant that is intended to be detected by the allele specific primer. Non-fluorescent AS-PCR was developed by Despite its simplicity, it would be ideal if the reaction and detection could be done in a single tube for each sample. To avoid setting up three separate reactions for each poly-T allele, fluorescent labeling of each allele-specific primer in a different color made visualization of each poly-T allele in a single tube reaction possible . As it iCFTR poly-T track analysis.In this report, we developed a simple and easy-to-implement standalone qualitative multiplex AS-PCR assay using standard length primers (less than 50 nucleotides). Our assay can be utilized as a reflex or standalone test for Genomic DNA (gDNA) samples from 115 individuals were included in this study. Of these samples, 60  were purIn-house gDNA from sample ID number S43 to S66, S89 and S90  were extxl instrument; Elim Biopharmaceuticals, Hayward, CA). Sequencing results indicated that NA11723 has 5T and 7T alleles with 12 and 10 TG repeats, respectively. NA13591 has 5T with 12 TG repeats and 9T with 10 TG repeats. Both 5T alleles contain the same number of TG repeats and would generate the same size AS-PCR products. A 5T plasmid clone from NA13591 was used in the study.Because there were no samples from the MUTCF-2 Cystic Fibrosis Mutation Panel that are homozygous for 5T alleles, we cloned 5T alleles from NA11723 and NA13591, two of samples from MUTCF-2 panel. A 267bp fragment was amplified using AmliTaq 360 DNA polymerase  and 250 nmol/L of each of the CFTR_ex9_P1 (5′-AAAACAAGCATCTATTGAAAATATCTGA-3′) and CFTR_ex9_P2 (5′-CTTGCCTGCTCCAGTGGAT-3′) primers. PCR products of each sample were cloned into pCRII-TOPO vector . Three individual clones from each of the samples, NA11723 and NA13591, were sequenced (ABI 3730CFTR (CGTTGTAAAACGACGGCCAGCCCCAAATCCCTGTTAAAAAATC-3′) for 7T, and CFTR_ex9_P16 (5′-ACCTAAATAGCTAAGCCCAAATCCCTGTTAAAAAAA ATC-3′) and CFTR_ex9_P16_SP6 (5′-GCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTAAGC CCAAATC-3′) for 9T. PCR product size for each allele is different, 88 to 98 bp for 5T, 108 to 118 bp for 7T and 130 to 140 bp for 9T, considering TG repeats at 5′ of the poly-T track range from 8 to 13.Three sets of primers were designed to identify 5T, 7T or 9T allele in the intron 8 of CFTR . All havThe multiplex AS-PCR is carried out in a 10-µL reaction containing 1X Melt Doctor HRM master mix , 25 ng of the Coriell, in-house manually extracted or 1 µL of the easyMAG extracted gDNA, and 750 nmol/L of CFTR_ex9_P1, 250 nmol/L of CFTR_ex9_P12 and CFTR_ex9_P16_SP6, 100 nmol/L of CFTR_ex9_P14 and 50 nmol/L of CFTR_ex9_P16. PCR was performed at 95 °C for 5 min, followed by 36 cycles at 95 °C for 15 s, 59 °C for 30 s and 72 °C for 45 s. An additional 72 °C for 5 min extension was added at the end of the 36-cycle reaction. PCR products were separated by size using the Qiaxcel DNA High Resolution cartridge and method OM800 on the Qiaxcel Advanced System (Qiagen) to determine whether samples contain 5T, 7T or 9T allele or a combination of any two of the alleles. For 5T plasmid, 170 pg of plasmid were added to the multiplex AS-PCR.After optimization of the multiplex AS-PCR, run-to-run and within-run reproducibility were evaluated using five gDNAs from MUTCF-2 panel, NA01531 (9/9T), NA07441 (7/9T), NA11277 (7/7T), NA11723 (5/7T) and NA13591 (5/9T), and a 5T plasmid clone as homozygous 5/5T. The final call of the poly-T alleles of each sample was compared to determine the reproducibility of the method. For 5T plasmid, 170 pg of plasmid were added to the multiplex AS-PCR.110 samples were tested to determine the length of the poly-T alleles using the newly developed multiplex AS-PCR. Among these samples, eighteen (sample ID S96 to S113) from MUTCF-2 of Coriell Cell Repositories were tested previously . Poly-T Sequences flanking the poly-T region of all the 92 samples of unknown poly-T alleles were amplified using primers CFTR_ex9_P1 and CFTR_ex9_P2 as in “Cloning of 5T Alleles”. Each PCR reaction contained 1X Melt Doctor HRM master mix, 250 nmol/ul of each of the primers and 50 ng of Coriell or in-house manually extracted or 2 µL of the easyMAG extracted gDNA. PCR products were run on Qiaxel to determine the proper amplification of a single 267 bp fragment. Then PCR products were treated with Exonuclease and Shrimp Alkaline Phosphatase based on manufacturer’s instruction . CFTR_ex9_P2 were used for Sanger sequencing . Poly-T status of each sample was determined from directly reading the sequencing chromatogram.CFTR . CFTR_ex9_P14, a 43-base primer for 7T includes 20 bases of M13 sequences added to the 5′ of the primer, generating fragment size of 108 to 118 bp. A 61-base or longer AS-PCR primer would be designed to generate the shortest 9T allele , that is at least 10-bp longer than the largest 7T allele . Technically, synthesis of such long primers is possible. The longest primer for Elucigene’s AS-PCR is 90 bases to detect 9T alleles  and for run-to-run reproducibility (three different runs on the same PCR instrument). All indicated that the multiplex AS-PCR was able to determine the poly-T alleles for all six poly-T allele combinations in the same run or different runs (data not shown).92 samples with unknown poly-T status were tested using the multiplex AS-PCR and run on Qiaxcel DNA High Resolution cartridge . After ACFTR. Our assay was able to detect all the combinations of the poly-T alleles, 5/5T, 7/7T, 9/9T, 5/7T, 5/9T, and 7/9T  on that instrument, we were able to obtain consistent results for poly-T testing. The main objective is to calibrate the annealing temperature on each instrument so that the specific target products are maximally amplified, whereas the non-specific by-products are minimized.Second, using a PCR instrument with good uniformity across all the wells would produce better reproducibility and more robust results. Most of PCR instruments are block-based and temperatures are known to vary among different wells of the same block . Such vaLastly, interpretation of the poly-T results needs to take the size range of each allele into consideration . With thAlthough the assay performed well in our hands, each lab would need to establish the protocol on its own instrument to implement the test. The first step would be to choose the PCR instrument with the best temperature uniformity in a laboratory. Instruments with multiple temperature sensors usually have better uniformity than a single sensor. Secondly, five of the 23 samples from MUTCF-23 panel with different poly-T alleles  would beCFTR. Our assay can be implemented as a reflex test in patients with positive R117H, if another method for CFTR mutation analysis didn’t include poly-T analysis. The assay can also be combined with any routine CFTR tests as a standalone method for molecular diagnosis of CBAVD. Furthermore, our AS-PCR design improves robustness and consistency for poly-T allele detection in CFTR.Overall, we developed a simple, easy to implement standalone multiplex AS-PCR method to detect all three poly-T alleles in intron 8 of 10.7717/peerj.468/supp-1Figure S1All the samples used for optimization of the multiplex AS-PCR have known poly-T allele information, NA01531 (9/9T), NA07441 (7/9T), NA11277 (7/7T), NA11723 (5/7T) and NA13591 (5/9T). 5T plasmid was cloned from NA11723 and NA13591. After Sanger sequencing of the plasmid clones, all the clones with 5T has 12 TG repeats, 7T allele from NA11723 has 10 TG repeats, and 9T allele from NA13591 has 10 TG repeats. 5 µl of the AS-PCR reactions were run on 4% NuSieve 3:1 agarose gel . NTC: No template control.Click here for additional data file.10.7717/peerj.468/supp-2Table S1Click here for additional data file.


Klebsiella pneumoniae (Kp) are of global concern from both clinical and public health standpoints. This retrospective study aimed to describe C-R Kp BSI epidemiology in a large teaching hospital in northern Italy.Bloodstream infections (BSI) due to carbapenem-resistant (C-R) Between 1 January 2007 and 31 December 2014, annual incidences both of C-R Kp BSI and of carbapenem-susceptible (C-S) Kp BSI were calculated as the number of events per 10,000 patient-days. A Chi square test for linear trend was used to assess the change in the incidence of C-R Kp BSI and C-S Kp BSI over the study period. Crude 30-day mortality rates were provided both for C-R Kp BSI and for C-S Kp BSI.p < 0.001). The highest incidence of C-R Kp BSI was observed in intensive care units (ICUs), with a peak of 22.01 C-R Kp BSI/10,000 patient-days in 2012. A less marked but significant increase of C-S Kp BSI was also observed (Chi square for trend p = 0.004). Crude 30-day mortality was 36.1 % in patients with C-R Kp BSI and 23.5 % in those with C-S Kp BSI.From 2007 to 2014, we observed 511 episodes of Kp BSI, 349 of which were caused by C-R Kp (68.3 %). The incidence of C-R Kp BSI considerably increased from 0.04/10,000 patient-days in 2007 to 1.77/10,000 patient-days in 2014 (Chi square for trend During the study period, we observed a dramatic increase in the incidence of C-R Kp BSI in our hospital. More concerted infection-control efforts are needed to contain this alarming C-R Kp diffusion. Klebsiella pneumoniae (Kp) are of global concern from both clinical and public health standpoints. Indeed, these severe infections are caused by rapidly spreading clones that are frequently resistant to almost all the antibiotics used in the everyday clinical practice  57–76). As many as 231/349 C-R Kp BSI occurred in males (66.2 %), thus defining a 2:1 male to female ratio. The median time to C-R Kp BSI diagnosis from hospital admission was 24 days (IQR 12–45), and the median length of hospital stay was 52 days (IQR 30–90).p < 0.001). Detailed year-by-year incidences of C-R Kp BSI are outlined in Table During the study period, the overall incidence of C-R Kp BSI was 0.92/10,000 patient-days, increasing from 0.04/10,000 patient-days in 2007 to 1.77/10,000 patient-days in 2014 (Chi square for trend p < 0.001). The singular trends of C-R Kp BSI and C-S Kp BSI over the study period are depicted in Fig. p = 0.004).Overall, the incidence of Kp BSI (C-R plus C-S) increased from 0.50/10,000 patient-days in 2007 to 2.42/10,000 patient-days in 2014 . However, this comparison was not further considered in our analyses, since it could not be adjusted for some important clinical confounders  that were not explored in this epidemiologic study.From 2007 to 2014 we noticed an impressive increase of more than 300 % in the incidence of Kp BSI in our hospital, that was mostly driven by C-R strains. This finding is of particular concern, since C-R Kp BSI carry a heavy burden in terms of morbidity and mortality .A worrisome and persistent increase of carbapenem-resistance among Kp isolates has been reported in Italy since 2005, with current national rates being as high as 34 % , 12. OurAll these measures have been already proven to reduce the incidence of infections due to multidrug resistant gram negatives, including C-R Kp –19. HoweThe present study has some important limitations. First, we could not depict the true pattern of C-R Kp diffusion, since only blood C-R Kp isolates were considered. However, C-R Kp BSI can provide a reliable picture of this diffusion, since blood cultures are collected from nearly all patients with C-R Kp BSI because of the symptomatic nature of the infection. Conversely, any possible estimation based on colonized patients would have been affected by an important selection bias due to the differences in the number of screened patients across the study years. Second, the retrospective nature of the study prevented us from investigating the degree of compliance to infection-control measures during the study period, as well as the molecular characteristics of C-R Kp isolates. In this regard, a major limitation is the lack of information about both the clonal type of Kp isolates and their mechanisms of resistance to carbapenems. Finally, this was a single center study, and incidences of C-R Kp BSI may differ significantly across centers. Therefore, any extrapolation of our findings to other settings should be made with due caution.In our hospital a dramatic increase in the incidence of C-R Kp BSI occurred from 2007 to 2014 that appeared to be only temporarily arrested by the intensification of hospital-based infection-control measures. More concerted efforts are necessary to tackle this alarming C-R Kp diffusion. Because of the dramatic shortage in antibiotics active against C-R Kp, further improvements in our infection-control protocols are also of paramount importance to curtail the high number of deaths due to C-R Kp BSI.


MTHFR gene were not associated with milk production traits from a breeding perspective. Single nucleotide polymorphisms (SNPs) at microRNA (miRNA) binding sites (miR-SNPs) can affect gene expression. This study aimed to identify the effects of miR-SNPs (g.2244A>G and g.2264A>G) in the caprine MTHFR 3' UTR on the milk production traits of dairy goats.5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism by irreversibly converting 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, a predominant circulating form of folate. Folate is reportedly important for milk protein synthesis, and MTHFR may be a key regulatory point of folate metabolism for milk protein synthesis in mammary epithelial cells. Prior to this study, polymorphisms of the n = 325) goats were used to detect SNPs in the caprine MTHFR 3' UTR by DNA sequencing. Two novel SNPs (g.2244A>G and g.2264A>G) were identified in the said region. The homozygous haplotype A-G of the SNPs g.2244A>G and g.2264A>G was significantly associated with milk yield and milk protein levels in GD goats (P < 0.05). Functional assays indicated that the MTHFR 2244 A → G substitution could increase the binding activity of hsa-miR-1266 with the MTHFR 3' UTR. The MTHFR 2264 A → G substitution could decrease the binding activity of hsa-miR-616 with the MTHFR 3' UTR. In addition, we observed a significant increase in the MTHFR mRNA levels of homozygous haplotype A-G carriers relative to those of homozygous haplotype G-A carriers. These results indicated that both SNPs altered the MTHFR mRNA levels. These altered levels of MTHFR mRNA may account for the association of SNPs with milk production traits.Guanzhong dairy (GD, This study is the first to report that the g.2244A>G and g.2264A>G polymorphisms were associated with milk production traits in GD goats. Further investigations should explore the underlying miRNA-mediated mechanisms that are modified by the g.2244A>G and g.2264A>G SNPs. The current study evaluated these SNPs as potential genetic markers in goats, with potential applications in breeding programs. MTHFR gene is located on chromosome 16 and has 13 exons spanning 14.639 kb .MTHFR 3'UTR, fragments of the MTHFR 3' UTR with different haplotypes  were amplified by PCR. The PCR products of the different haplotypes  were isolated and separated by agarose gel electrophoresis. These products were then linked to a pMD19-T vector with a TA Cloning Kit . The recombinant pMD19-T vectors with different haplotypes were digested by the XhoI and NotI endonuclease enzymes. Finally, the digested products with different haplotypes were inserted between the Renilla and firefly luciferase genes in a psiCHECK-2 vector .The plasmids containing the A-G, G-A, A-A and G-G haplotypes were obtained and then confirmed by sequencing. For the luciferase reporter assay, GMECs were placed in 12-well plates (5 × 105 cells per well) and then cotransfected with psiCHECK-2 vectors containing the 3'UTR-haplotypes A-G, G-A, A-A and G-G. The mimics of miRNAs  and their negative controls  were cotransfected with the reporter plasmids to a final concentration of 20 nmol/μL. At 36 h after transfection in GMECs, Renilla luciferase activity in lysates was measured with the Dual-Luciferase Reporter Assay System .The results were normalised against firefly luciferase activity. Assays were performed according to the manufacturer’s suggestions. Each experiment was independently performed three times, and each sample was evaluated in triplicate. The Mann–Whitney test was used to compare data, and a two-sided P value of 0.05 was considered significant.To construct the luciferase reporter plasmids containing the MTHFR 3'UTRby DNA sequencing . Four alleles of the g.2244A>G and g.2264A>G SNPs introduced several different miRNA sites (P < 0.05)  were identified in the caprine ing Figs  and 2. TNA sites . The PICNA sites . The genP < 0.05; Table D in P < 0.05). The combined effects of the two SNPs (g.2244A>G and g.2264A>G) showed that individuals with the AA-GG (homozygous haplotype A-G) genotype had higher milk yield than individuals with the AA-AA (homozygous haplotype A-A), GG-AA (homozygous haplotype G-A), GG-GG (homozygous haplotype G-G) and AG-AG genotypes .Individual GD goats with AA and AG genotypes had greater milk yield than those with the GG genotype at the g.2244A>G locus , but the difference with the haplotype 2244A-2264G was not significant . However, hsa-miR-505 did not significantly affect luciferase expression  is a potential candidate SNP that modulates miRNAs; the MTHFR mRNA complex has the greatest change in the binding free energy for hsa-miR-149; based on luciferase analysis, hsa-miR-149 inhibited the activity of the reporter vector carrying the T allele, but not the G allele [MTHFR on animal production traits have remained largely unknown. In the present study, the results of our integrative analysis showed that the g.2244A>G and g.2264A>G SNPs may change the binding activity of miRNAs (hsa-miR-1266 and hsa-miR-616) with the MTHFR 3'UTR. We used a bioinformatics analysis method similar to that of Sun et al. [MTHFR gene.The 3'UTR polymorphisms may be within or at the vicinity of the miRNA binding site; these polymorphisms may interfere with miRNA function and lead to differential gene expression, thereby affecting the animal phenotype . Previoupression , 24. TheG allele . The effMTHFR 2244 A → G substitution could increase the binding activity of hsa-miR-1266 with the MTHFR 3'UTR, whereas the MTHFR 2264 A → G substitution could decrease the binding activity of hsa-miR-616 with the MTHFR3'UTR. These data were consistent with results of in silico analysis, thereby suggesting the functional interaction between hsa-miR-1266 or hsa-miR-616 and the MTHFR mRNA 3' UTR. We further examined the MTHFR mRNA levels in GD goats with different haplotypes. A significant increase in the MTHFR mRNA levels of the homozygous haplotype A-G carriers was observed relative to those of the homozygous haplotype G-A carriers. These findings consistently suggested that the g.2244A>G and g.2264A>G SNPs altered the MTHFR expression level, and they were associated with milk production traits. However, this study had several limitations. Firstly, the implied regulation of MTHFR expression by hsa-miR-1266 and hsa-miR-616 is based on preliminary results. Further functional experiments are required to analyse the effects of this polymorphism on MTHFR function. Secondly, the influence of other miRNAs on mutant forms of the MTHFR gene must be considered.The results of the association analysis showed that individuals with the homozygous haplotype A-G had higher milk yield and protein content than those with other haplotypes. Functional assays indicated that the In conclusion, our data suggested that the g.2244A>G and g.2264A>G polymorphisms were associated with milk production traits in GD goats. Future investigations should explore the underlying miRNA-mediated mechanisms that are modified by the SNPs g.2244A>G and g.2264A>G. This study evaluated these SNPs as genetic markers for goat genetics and breeding, with potential applications in breeding programs.S1 FigThe green boxes represent potential miRNA target sites.(TIF)Click here for additional data file.S2 FigMTHFR 3THFR.Green represents miRNA. Red represents (TIF)Click here for additional data file.S1 FileMTHFR gene (Table A). Primer information for real-time quantitative PCR (Table B). Genotypic distribution and allelic frequencies of two SNP loci in the caprine MTHFR gene (Table C). Association analysis of g.2244A>G and g.2264A>G loci with milk production traits (means ± standard errors) in Guanzhong dairy goats (Table D). Luciferase activity (means ± standard deviation) of the psiCHECK-2-MTHFR 3′UTR vector that include haplotypes 2244G-2264A and 2244A-2264G (Table E). Luciferase activity (means ± standard deviation) of the psiCHECK-2-MTHFR 3′UTR vector that include haplotypes 2264G-2244G and 2264A-2244A (Table F). Comparison of expression levels of caprine MTHFR mRNA in mammary glands among four haplotypes (Table G).Primer information of (DOC)Click here for additional data file.


O-glycosylated and O-glycosylated forms of the mucin identified three major intracellular populations of MUC5B . Biophysical analysis of recombinant MUC5B COOH-terminus  expressed in 293-EBNA cells showed that MUC5B dimerizes by disulfide linkage. Pulse-chase studies in the HBEC ALI cultures showed that non-O-glycosylated MUC5B was synthesized within 20 min of metabolic labeling and O-glycosylated, polymeric mucin within 2 h. Radiolabeled O-glycosylated mucin polymers were secreted within 2 h and the majority were released by 48 h. These data indicate that MUC5B follows a similar assembly to the related glycoprotein, von Willebrand factor (vWF); however, unlike vWF the MUC5B polypeptide shows no evidence of major proteolytic processing of D-domains during the production of the mature secreted polymeric mucin in normal and cystic fibrosis (CF) primary bronchial epithelial cells. In contrast, MUC5B D-domains were modified by neutrophil elastase, a protease commonly found in CF sputum, demonstrating that proteolytic degradation of MUC5B is an extracellular event in CF sputum. These results define the pathway for synthesis of MUC5B in primary human goblet cells.MUC5B is a major polymeric mucin in the airway mucus gel and is an essential component of innate defense of the respiratory epithelium. Knowledge of the synthesis and intracellular processing of MUC5B is incomplete. We investigated the molecular details of MUC5B assembly in primary human bronchial epithelial cells (HBECs) grown at an air-liquid interface (ALI). Electrophoretic and centrifugal separations of intracellular forms of MUC5B probed with antibodies specific for non- This glycoconjugate-rich layer is an important component of innate defense and is vital for normal physiology. In the airways the properties of the gel are tailored for transport, and, in conjunction with the ciliated epithelium, mucus functions to remove pathogens, particulates, and toxins from the lung. However, accumulation of mucus, with suboptimal transport properties, is a pathological feature of airway diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease, and asthma   , 34. Alt domains .2-terminus  the time course of MUC5B intracellular assembly, 2) the role of COOH-terminal protein domains in the assembly process, and 3) whether proteolytic processing was a feature of MUC5B assembly, packaging, or expansion in normal and CF cells.To gain insight into the molecular detail of MUC5B biosynthesis we investigated Normal HBECs (donor numbers 7152 and 2F1578) grown at the University of Manchester were purchased from BioWhittaker  and grown by methods based on those of Gray and coworkers . At confSimilar bronchial epithelial cell cultures derived from the lungs of CF patients (CF BECs) were grown at the University of North Carolina (UNC). The cells were derived from lungs obtained at transplantation under UNC Institutional Review Board-approved protocols consistent with all US federal and institutional requirements for informed consent and confidentiality. The cells were grown on the same Transwell-COL inserts as above, using similar cell culture protocols and media prepared in the UNC CF Research Center Cell Culture facility , 5, 17.N′-ethylmaleimide (lysis buffer). Before lysis, cells were washed (as above) and then transferred to wells containing PBS, to wash the underside of the inserts (24 mm), and subsequently transferred to wells containing 0.5 ml lysis buffer basally and 1 ml apically, and plates were then gently shaken overnight at 4°C. Lysates were analyzed by Western blotting after agarose gel electrophoresis and fractionated by density gradient centrifugation . Fractions from the density gradient were analyzed by immunodetection after slot blotting.At least once a week post-ALI formation, the apical surface of the cells was washed gently with 0.5 ml prewarmed PBS (one quick wash plus one 30-min wash). These washes were pooled (from 7–24 days post-ALI) and stored at 4°C; this pool is then referred to as apical secretions. Cells were lysed by the addition of 6 M guanidinium chloride (GdmCl), 5 mM 2. Inserts were then transferred to wells containing methionine-free ALI media with [35S]l-methionine [3 MBq (83 μCi)/24 mm insert] and pulse labeled for 15, 20, or 30 min (stated in legend to appropriate figure) at 37°C, 5% CO2. At chase time, t = 0 inserts were again transferred to prewarmed PBS, twice, then into 10 × methionine ALI media and incubated between 10 min and 120 h at 37°C and 5% CO2. At given time points the apical surface of the cells was washed twice with PBS (one quick wash and then one 5-min wash). If the time point was more than 8 h, three washes were performed in the final hour. At each time point cells were lysed as above and lysates were dialyzed into 6 M urea, for electrophoresis, or detergent-free immunoprecipitation (IP) buffer  for immunoprecipitation.HBECs were used for pulse-chase experiments between 15 and 25 days post-ALI formation, because this has been found to be the period in which mucin production is at its greatest . The apiPBS washes of the apical surface of the cultures were pooled and GdmCl was added to 4 M, mixed overnight at 4°C, to ensure solubilization of all the mucins, and then dialyzed into 6 M urea. Radiolabeled molecules were separated by agarose gel electrophoresis, transferred to nitrocellulose, and subsequently detected by phosphoimaging .Cell lysates or apical washes were dialyzed into IP buffer; Triton X-100 was added to a final concentration of 1%, then mixed with Protein-A Sepharose CL-4B (10% wt/vol). After 1 h of mixing at 4°C, the solution was centrifuged and the resulting supernatant was mixed with fresh Protein A-Sepharose containing 1:100 LUM5B-13 and incubated with rotation at 4°C for 48–65 h. Three detergent-free IP buffer washes preceded elution of the bound molecules with two washes of 6 M GdmCl. The eluted solution was then subject to dialysis into 6 M urea before electrophoresis.Briefly, samples were run on 0.7% agarose gels (wt/vol)  for 16 h at room temperature. Where unreduced samples were run, gels were treated with reducing agent  prior to transfer. Transfer of mucins to nitrocellulose was via a vacuum blotter at 45 mbar for 1 h 30 min . The datQ9HC84) COOH-terminal construct was created consisting of D4-B-C-CK  staining and MUC5B by use of immunodetection after slot blotting onto nitrocellulose . Mucin-cMUC5B (100 μg/ml) was incubated with or without 1 μg/ml neutrophil elastase  in PBS fO-glycosylated and non-O-glycosylated MUC5AC) (O-glycosylated MUC5AC) (O-glycosylated and non-O-glycosylated MUC5B)  raised against the synthetic peptide TPSSTPGTTWILTC was used at 1:1,000 for immuno- and Western blots. Band intensities were quantitated by use of Quantity One software and GS-800 scanner .MUC5AC [polyclonal antiserum MAN-5ACI (anti- MUC5AC) ] was use MUC5AC)  was usedd MUC5B) ] was useO-glycosylated MUC5B, specifically recognizes the non-O-glycosylated mucin (O-glycosylated and O-glycosylated MUC5B (O-glycosylated MUC5B migrated as two bands with high electrophoretic mobility (lane 1). After reduction, a single band was observed that migrated further into the gel (lane 5). The non-O-glycosylated forms of MUC5B likely represent 1) a disulfide-linked multimer, probably native polypeptide dimer , 2) native polypeptide monomer ; and 3) reduced polypeptide monomer . Importantly, the probe for non-O-glycosylated MUC5B identified bands that were distinct from those detected by Mab2011 (lanes 2 and 6), a monoclonal antibody specific for non-O-glycosylated MUC5AC, the other polymeric mucin produced by these cells in culture . After treatment with DTT, the resultant reduced, O-glycosylated monomer exhibited a marked increase in electrophoretic mobility . Neither native nor reduced O-glycosylated mucins were reactive with LUM5B-13, the non-O-glycosylated mucin probe (lanes 1 and 5), even after overexposure of the blots. Furthermore, under normal conditions, none of the MUC5B in the apical secretions from the cells was reactive with LUM5B-13 (data not shown). It is noteworthy that the O-glycosylated MUC5B is indistinguishable by electrophoresis from MUC5AC, either at the polymeric mucin level (lanes 3 and 4) or as reduced O-glycosylated monomers (lanes 7 and 8).In contrast to the non-O-glycosylated mucins (high buoyant density), from non-O-glycosylated mucins (low buoyant density) (O-glycosylated MUC5B (1.38–1.5 g/ml) from the non-O-glycosylated polypeptide (1.25–1.32 g/ml)  , 25. Slo32 g/ml) C.O-glycosylated form of MUC5B, whereas LUM-5B13 detected only the polypeptide precursors. The MAN-5BI data indicated that O-glycosylated MUC5B was the major form of the mucin within the cells. Furthermore, it is important to note that MUC5B is the predominant mucin produced by this culture model , and the lysates were subjected to immunoprecipitation with LUM5B-13. Agarose gel electrophoresis of the immunoprecipitates showed transfer of radioactivity from non-O-glycosylated monomers (NPM) to dimers (NPD) over the 2 h chase period  was visible inside the cells after 2 h postlabel . This change in mobility, after treatment with DTT, is consistent with the change in electrophoretic properties of polymeric O-glycosylated MUC5B shown in O-glycosylated mucin polypeptide probe LUM5B-13 (data not shown). Taken together these data confirmed that the radiolabeled molecules found at the top of the gel were O-glycosylated mucin polymers. However, it is important to reiterate that we cannot distinguish between MUC5B and the other polymeric mucin produced by these cells, MUC5AC, since both mucins had similar electrophoretic properties and neither of the mucin antisera (MAN-5BI and MAN-5ACI) were able to quantitatively immunoprecipitate the O-glycosylated mucins.Further pulse-chase studies were performed to analyze the synthesis of UC5B see B; lane 3ostlabel A and remostlabel B and theO-glycosylated polymers over a longer time scale, the experiment was repeated with increased chase times . The results showed that the majority of the radiolabeled mucins were released from the cells at baseline by 72–96 h  remains associated with the mature polymer, via a calcium-mediated interaction with the D3 domain, until after secretion when the change in environment (increased pH and decreased Ca2+) cause it to dissociate and the polymer unfurls  were subjected to trypsin digestion followed by tandem MS to identify the major proteins present. There were no peptides identified from MUC5B in the low buoyant density fraction. In contrast, in the high-buoyant density mucin-rich fraction, 48 peptides were identified from MUC5B, which were distributed throughout the molecule (data not shown). These results showed no evidence for the absence of NH2- (or COOH)-terminal protein domains from MUC5B and suggested that, if the polypeptide of MUC5B had been cleaved, then the fragment(s) had remained attached to the polymer. This is not unlikely because the terminal regions of MUC5B contain multiple intramolecular disulfide linkages and polypeptide cleavage will not necessarily generate “free” fragments. However, this possibility was ruled out by subsequent experiments in which the mucins were reduced and carboxymethylated and resubjected to CsCl/4 M GdmCl density gradient centrifugation (fractions 1-5) and analysis of the high-density reduced mucin fraction (fractions 7-18) revealed 32 peptides across the NH2-terminus of the MUC5B polypeptide  compared with untreated MUC5B . These results support that proteolytic cleavage of MUC5B reported in CF is an extracellular event in sputum.Proteolytic cleavage of MUC5B has been reported in CF , 9; to aypeptide . These rypeptide  for 4 anMUC5B is a major polymeric mucin in airway mucus . Here weO-glycosylated and non-O-glycosylated MUC5B. Using this approach we have shown that O-glycosylated MUC5B is the major form of the mucin within the cells and, importantly, that O-glycosylated MUC5B (not non-O-glycosylated) is secreted from cells. Thus O-glycosylation of MUC5B is likely to be a prerequisite for passage through the secretory pathway; however, experimental verification is necessary to test the observation.We were able to distinguish on basis of buoyant density and electrophoretic mobility between 2-terminal multimerization via disulfide linkage between D3 domains  and disulfide-linked D3 dimers that has been proposed to organize vWF polymers for storage within Weibel-Palade bodies  and The Wellcome Trust (065833/Z/01/Z). The Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, is supported by core funding from the Wellcome Trust [088785/Z/09/Z].This work was supported by grants from No conflicts of interest, financial or otherwise, are declared by the author(s).C.R., S.K., and S.J.W. performed experiments; C.R. and S.K. prepared figures; C.R., S.K., and D.J.T. drafted manuscript; C.R., C.W.D., P.W., and D.J.T. edited and revised manuscript; C.R. and D.J.T. approved final version of manuscript; S.K., S.J.W., and D.J.T. analyzed data; C.W.D., P.W., and D.J.T. interpreted results of experiments; P.W. and D.J.T. conception and design of research.


Mycobacterium tuberculosis strains for the selected antigen, and the capability to induce robust T-cell responses in the majority of human populations. A tool in the identification of putative T-cell epitopes is in silico prediction of major histocompatibility complex (MHC)-peptide binding. Candidate TB vaccine antigen Mtb72F and its successor M72 are recombinant fusion proteins derived from Mtb32A and Mtb39A . Adjuvanted Mtb72F and M72 candidate vaccines were shown to induce CD4+ T-cell responses in European, US, African and Asian populations.Requisites for an efficacious tuberculosis (TB) vaccine are a minimal genomic diversity among infectious Mycobacterium strains causing human TB disease, and H37Ra) was assessed by multiple alignments using ClustalX. For Mtb32A, Mtb39A and Mtb72F, 15-mer human leukocyte antigen (HLA)-class II-binding peptides were predicted for 158 DRB1 alleles prevailing in populations with high TB burden, 6 DRB3/4/5, 8 DQ and 6 DP alleles, using NetMHCII-pan-3.0. Results for 3 DRB1 alleles were compared with previously published allele-matched in vitro binding data. Additional analyses were done for M72. Nonameric MHC class I-binding peptides in Mtb72F were predicted for three alleles representative of class I supertypes A02, A03 and B07, using seven prediction algorithms.Sequence conservation of Mtb32A, Mtb39A, Mtb72F and M72 among 46 strains  contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) disease. Requisites for an efficacious TB vaccine to be used in populations world-wide are a minimal genomic variation of the target antigen among different Mtb strains, and the ability of the antigen’s contents of peptides to bind to the largest possible subset of HLA alleles in the host populations. The TB vaccine should also be able to induce robust T-cell responses ), are major determinants for human vaccine efficacy. esponses . While Cin silico prediction of this binding has become a tool in vaccine design. Predictive computer algorithms are generally trained by databases of experimentally validated epitopes with T-cell stimulation potential, such as SYFPEITHI  was below 50 nM or as a ‘weak binder’ if the IC50 was >50 nM but <500 nM. The total numbers of epitopes shown in this study represent the sum of the strong and the weak binders.HLA-II binding predictions were generated for 15-mer peptides (overlapping by 14 amino acids) of Mtb32A, Mtb39A and Mtb72F using NetMHCIIpan-3.0 , and for alleles , using tMHC-I-binding predictions for the A*02:01, A*03:01 and B*07:02 alleles were done for 9-mer peptides in Mtb72F using NetCTLpan-1 , NetMHCpThe data sets supporting the results of this article are included in Tables The authors declare that this study does not involve any ethical issues, since no animal or human experiments were performed as part of the research for this manuscript.


It is generally accepted that nitric oxide (NO) or its derivatives, reactive nitrogen species (RNS), are involved in the development of Parkinson’s disease (PD). Recently, emerging evidence in the study of PD has indicated that protein S-nitrosylation triggers the signaling changes in neurons. In this study, SH-SY5Y cells treated with rotenone were used as a model of neuronal death in PD. The treated cells underwent significant apoptosis, which was accompanied by an increase in intracellular NO in a rotenone dose-dependent manner. The CyDye switch approach was employed to screen for changes in S-nitrosylated (SNO) proteins in response to the rotenone treatment. Seven proteins with increased S-nitrosylation were identified in the treated SH-SY5Y cells, which included proliferating cell nuclear antigen (PCNA). Although PCNA is generally located in the nucleus and participates in DNA replication and repair, significant PCNA was identified in the SH-SY5Y cytosol. Using immunoprecipitation and pull-down approaches, PCNA was found to interact with caspase-9; using mass spectrometry, the two cysteine residues PCNA-Cys81 and -Cys162 were identified as candidate S-nitrosylated residues. In addition, the evidence obtained from in vitro and the cell model studies indicated that the S-nitrosylation of PCNA-Cys81 affected the interaction between PCNA and caspase-9. Furthermore, the interaction of PCNA and caspase-9 partially blocked caspase-9 activation, indicating that the S-nitrosylation of cytosolic PCNA may be a mediator of the apoptotic pathway. Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by a massive and specific loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) , 2. The -) have been observed in PD patients Chronic lspases-3  at the aspases-3 , whereasspases-3 . AccordiIn this research, we used rotenone-treated SH-SY5Y cells as a typical PD model to investigate the mechanism between SNO proteins and neuronal apoptosis. The canonical approach of SNO proteins detection is biotin switch technique, from which many proteomic strategies were derived for S-nitrosoproteome analysis . Herein,G-Methyl-L-arginine acetate salt (L-NMMA), S-Methyl methanethiosulfonate (MMTS), Sodium L-ascorbate, Neocuproine and DMF were purchased from Sigma-Aldrich. 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FM DA) were obtained from Molecular Probes. Biotin-HPDP and NeutrAvidin agarose were obtained from Pierce Biotechnology. CyDye DIGE Fluors were obtained from GE Healthcare. Rabbit anti-PCNA polyclonal antibody was obtained from Beijing Protein Innovation, Ltd.; rabbit anti-nNOS and goat anti-biotin were obtained from Cell Signaling Technology. All other antibodies used in this study were purchased from Santa Cruz Biotechnology.Rotenone, N2 atmosphere.Human neuroblastoma SH-SY5Y and HeLa cervix carcinoma cell lines were obtained from Type Culture Collection (China Center). SH-SY5Y and HeLa cells were cultured in DMEM/F12 and DMEM (Gibco), respectively, and supplemented with 10% (v/v) fetal bovine serum, 0.1 U/ml penicillin, and 0.1 mg/ml streptomycin (PAA) at 37°C in a humidified 5% COThe SH-SY5Y cells were transferred to a low serum medium that contained 0.5% FBS for 12 h prior to further treatment. The rotenone solution was freshly prepared prior to each experiment in dimethyl sulfoxide (DMSO), and DMSO served as the vehicle control. L-NMMA, a NOS inhibitor, was dissolved in deionized water and added to the culture media at the final concentration of 0.5 mM 4 h prior to rotenone treatment. The final volume ratio of vehicle or drug solution to the media was controlled within 0.05%.ex = 495 nm and λem = 515 nm using a Tecan Safire5 microplate reader.S-Nitrosocysteine (SNOC) was used to generate NO according to previous methods  and was The protein samples were dissolved in lysis buffer that contained 50 mM Tris-HCl, pH 7.4, 2% SDS, 10 mM dithiothreitol (DTT), and 5 mM EDTA with supplements of proteinase inhibitors. The denatured proteins were separated on a 12% SDS—PAGE, and the separated proteins were transferred onto a polyvinylidene difluoride membrane. After blocking and washing, the membrane was incubated with the indicated primary antibody followed by incubation with horseradish-linked secondary antibody. The immuno-recognition signals were detected by chemiluminescence using ImageQuant ECL , and the chemiluminescence was quantified using PDQuest software version 8.0.1.6 cells/mL, and incubated with 5 μL annexin V-FITC and 10 μL PI for 15 min in the dark at room temperature. Then, these cells were added into 400 μL binding buffer and measured by FACSCalibur flow cytometer (Becton Dickinson).SH-SY5Y cells treated with or without 500nM rotenone for 16h were trypsinized and the cell pellets were collected. The cells were resuspended in 100 μL binding buffer at 1 × 10The biotin switch technique (BST) was performed according to a previous report . Cell lyex = 548 nm and λem = 560 nm for the Cy3-labeled sample and λex = 641 nm and λem = 660 nm for the Cy5-labeled sample, and the relative fluorescence intensity for a 2DE spot was estimated by the ratio of Cy5 against Cy3. Inversely, on the other 2DE running, the rotenone sample labeled with Cy5 was the SH-SY5Y cell treated with rotenone, while the internal standard sample labeled with Cy3 was a mixture of equal quantities of the control and rotenone sample. The sample loading and spot intensity estimation were the same as above. Since the composition in the internal standard sample was consistent, the fold-change for each spot could be obtained by comparison of the relative fluorescence intensity. The florescence images were analyzed using PDQuest software version 8.0.1. All the CyDye switch/2D-DIGE experiment, the related proteomic analysis and protein identification were conducted in three independent experiments.The combination of CyDye switch and DIGE was used to enrich and separate the SNO proteins in the SH-SY5Y cells. The cell lysates were incubated with 4X volume blocking buffer that contained 250mM HEPES-NaOH, pH7.4, 1mM EDTA, 0.1mM Neocuproine, 2.5% SDS, and 20mM MMTS, and the incubation was performed at 50°C for 30 min in the dark with vortex every 5 min. After the free thiols of the cell lysates were blocked and the S-nitrosothiols were reduced, the excess ascorbate was removed by acetone precipitation, and the precipitated proteins were dissolved in CyDye labeling buffer, which contained 30 mM Tris-HCl, 7 M urea, and 4% CHAPS, pH 8.0. CyDye DIGE Fluor dyes were used to label proteins at the final concentration of 10 μM. The labeling reaction was quenched by 2X CyDye labeling buffer that contained 2% v/v IPG buffer for pH 3–10 and 130 mM DTT. In the CyDye switch/2D-DIGE experiment, inverse strategy was adopted. On one 2DE running, the control sample labeled with Cy5 was the SH-SY5Y cell without rotenone treatment, while the internal standard sample labeled with Cy3 was a mixture, in which equal quantities of the protein lysates from the SH-SY5Y cells treated with and without rotenone were well mixed. The pooling of the equal quantities of the control sample and the internal standard sample was loaded to electrophoresis. Following 2DE, the gel was scanned by a Molecular Imager PharosFX System (Bio-Rad) with λ−1, 190°C; capillary, −5,500 V ; end plate offset, −500 V; funnel 1 RF, 200 Vpp; funnel 2 RF, 200 Vpp; in-source CID energy, 0 V; hexapole RF, 100 Vpp; quadrupole ion energy, 5 eV; collision gas, argon; collision energy, 10 eV; collision RF 200/400 Vpp (timing 50/50); transfer time, 70 μs; prepulse storage, 5 μs; pulse frequency, 10 kHz; and spectra rate, 3 Hz. The MS/MS spectra data were searched against the SwissProt database using Data Analysis software  and the MASCOT in-house search engine (MatrixScience).The selected 2D-DIGE spots were collected and subjected to in-gel digestion with trypsin following the protocol previously described . The pepTo identify the proteins in the PCNA interactome, immunoprecipitates from SH-SY5Y cells were subjected to gel-assisted digestion as previously described . The tryPCNA subcellular localization in the SH-SY5Y or HeLa cells was observed with a standard immunofluorescence staining protocol . Cells o2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 0.1 mM PMSF, and protease inhibitors. The resuspended cells were broken by passing several times through a G26 needle. The homogenate was centrifuged at 1000 g for 15 min to precipitate the nuclear fraction, and the supernatant was further centrifuged at 105 g for 30 min in L-100 XP ultracentrifuge (Beckman). The resulting supernatant was used as the cytosol fraction.The cell pellet was washed twice with ice-cold PBS and resuspended in 5X volume of ice-cold isolation buffer containing 20 mM Hepes—KOH, pH 7.5, 10 mM KCl, 1.5 mM MgClThe SH-SY5Y cytosol was kept in isolation buffer for 30 min at 4°C. Following centrifugation at 12,000 g for 20 min, the supernatants were incubated with anti-PCNA antibody to immunoprecipitate PCNA-interacting proteins. The eluted immunoprecipitates were identified by LC-MS/MS or loaded onto 12% SDS-PAGE gels followed by Western blotting using anti-caspase-9 antibody as the primary antibody.Escherichia coli BL21 (DE3) cells. The His-tagged fusion proteins were purified with nickel-agarose beads (QIAGEN) according to the manufacturer’s instructions. Approximately 50 μg His-PCNA protein were incubated overnight at 4°C with the lysates obtained from HeLa. Following incubation, nickel-agarose beads were added to capture the recombinant protein and the proteins that bound to it. The PCNA-interacting proteins were identified by Western blot.Using the pET system as indicated, wild-type or mutated recombinant His-PCNA proteins were expressed in The wild-type PCNA sequence was amplified by PCR, using 5’-CGCGGATCCATGTTCGAGGCGCGCCTG-3’ (forward) and 5’-CCGCTCGAGCTAAGATCCTTCTTCATCCTCGATC-3’ (reverse) primers, from the cDNA of the human hepatocarcinoma cell line Huh7 and was inserted into the expression vector pET-28a. Mutant PCNA plasmids were constructed using the mutated PCR primer pairs for C81A, 5’-GTCCAAAATACTAAAAGCCGCCGGCAATGAAGATATC-3’ (forward) and 5’-GATATCTTCATTGCCGGCGGCTTTTAGTATTTTGGAC-3’ (reverse), and for C162A, 5’-GCTGTTGTAATTTCCGCAGCAAAAGACGGAGTG-3’ (forward) and 5’-CACTCCGTCTTTTGCTGCGGAAATTACAACAGC-3’ (reverse). The double-mutant C81A/C162A plasmid was also constructed with the same mutated PCR primers. The mutated PCNAs were inserted into pET-28a for protein expression.HeLa cytosol was incubated with cytochrome c , 1 mM dATP and the indicated exogenous PCNA for 30 min at 37°C. The cleavage of caspase-9 was detected by Western blot using a polyclonal antibody against caspase-9 .The PCR product of PCNA gene with cloning sites of BamH1 and Xho1 was inserted into pcDNA3.1(+) plasmid (Invitrogen) for generation of transfection vector and the pcDNA3.1-PCNA was transfected to SH-SY5Y cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.t test.All experimental data were generated in triplicate (at minimum). The data are presented as the mean ± SEM, and significant differences among the test groups were determined by Student’s According to Fang et al., treatment with rotenone increases the NO content in SH-SY5Y cells, resulting in events that pathologically mimic the apoptosis of dopaminergic neurons in PD . We follCyDye switch/2D-DIGE is a common approach to analyze S-nitrosoproteomes –41; in tNext, the BST was performed. Its specificity in SH-SY5Y cells was demonstrated . WesternPCNA, a processivity factor for DNA polymerase ε in eukaryotic cells, is predominately located in the nucleus and involved in the apoptotic pathway. Recently, PCNA was also reported to be located in the cytosol of leukocytes . We inveHow does cytosolic PCNA perform its biological functions in SH-SY5Y cells, especially under NO stress? To answer this question, we searched for proteins that interact with PCNA. CoIP with antibody against PCNA was conducted using the cytosolic fraction of SH-SY5Y cells, and the proteins that interacted with cytosolic PCNA were identified by LC-MS/MS. As shown in There are 6 cysteine residues in PCNA. Which cysteine residue(s) of PCNA is sensitive to NO stress? Considering that the amount of native PCNA immunoprecipitated from SH-SY5Y cells is rather limited, we generated a full-length recombinant PCNA to identify its S-nitrosylation site(s) in vitro. After the recombinant PCNA was incubated with SNOC, its S-nitrosylation status was examined using BST/Western blot. The results obtained via MS/MS were further verified by site-directed mutagenesis experiments. The Cys81 or Cys162 residue was substituted with alanine to yield recombinant PCNA mutants. The WT or mutant forms of PCNA were immobilized on nickel beads, exposed to SNOC or control conditions, and then incubated in the dark with a cytosolic fraction of HeLa cells, in which no PCNA was constitutively expressed. The amount of caspase-9 in the pull down was detected by Western blotting. As shown in We next considered whether the S-nitrosylation of Cys81 of PCNA occurs in SH-SY5Y cells. We employed BST in SH-SY5Y cells with and without rotenone treatment and labeled the SNO proteins with biotin-HPDP. The proteins were run on an SDS-PAGE gel, and the band that corresponded to the PCNA molecular mass was excised and in-gel digested by trypsin, followed by peptide identification with LC-MS/MS. The spectrum count was used to evaluate the protein abundance. Compared with SH-SY5Y cells without rotenone treatment, the percentage of the peptides that contained S-nitrosylated Cys81 was increased following rotenone administration . Thus, tCaspase-9 activity can be regulated in many ways, such as phosphorylation by kinases or blockage by heat shock proteins. The activity of caspase-9 cleavage was evaluated using a HeLa cytosolic fraction, which contained apoptosome components, such as caspase-9 and APAF-1 . In thisIt is generally accepted that a combination of genetic susceptibilities and environmental factors cause PD pathogenesis. In previous decades, several hypotheses for the selective neurodegeneration upon oxidative stress have been proposed to explain the relevant molecular mechanisms. ROS, the main free radicals, likely play an important role in PD, including the abnormalities of iron metabolism and protein modification under oxidative stress in the PD brain. NO is another important free radical species that were widely observed in PD animal models and PD patients. For instance, in Sprague-Dawley rats injected with rotenone, Yao et al. observed that nitrosative stress was augmented which led to increased parkin S-nitrosylation ; in miceHerein, using the CyDye switch approach, we have successfully detected approximately 200 proteins that were likely S-nitrosylated in SH-SY5Y cells, including 7 proteins that showed increased S-nitrosylation in response to the increased NO stress induced by rotenone. Of these SNO proteins that are sensitive to NO stress, most have been observed as the S-nitrosylated adducts in previous studies. Protein DJ-1, a causative gene for familial PD, is a multi-functional protein involved in transcriptional regulation, molecular chaperoning and cell survival antagonization. Cys46 and Cys53 of DJ-1 are susceptible to S-nitrosylation; Cys46, which participates in the dimerization of DJ-1, is particularly susceptible . ThioredPCNA was recently identified in the cytosol of several leukocytes and was determined to be an apoptotic regulator through its interaction with caspases . The manThe activation of caspase-9 is an initial event in the intrinsic apoptosis pathway. It is generally accepted that the activation requires binding of its precursor, procaspase-9, to a complex of two proteins, APAF-1 and cytochrome c, and the self-cleavage specific aspartic residues of procaspase-9. Cleaved caspase-9 further processes other caspase members, including caspase-3 and caspase-7, which leads to the initiation of the apoptotic pathway. However, the activation of caspase-9 is restricted within the triple protein complex and mediated by several protein components or posttranslational modifications, such as HBXIP , TUCAN , ERK2 6, and PKAS1 FigSH-SY5Y cells in the low-serum media were pretreated with L-NMMA followed by the addition of rotenone. The levels of nNOS under the different treatments were detected by Western blot.(TIF)Click here for additional data file.S2 FigThe lysate of SH-SY5Y cells with and without rotenone treatment was obtained and subjected to BST. The biotinylated proteins were pulled down with NeutrAvidin beads and detected by Western blot using anti-caspase-9.(TIF)Click here for additional data file.S3 FigδC represents biotin-HPDP derivatized cysteine (+428), which was included in the b- or y-ion series.(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.


Bradyrhizobium, which is one of the ecologically important bacterial genera associated with plants. We discuss the methodology of phylogenetic assignments of plant-associated bacteria by 16S rRNA pyrosequence.Pyrosequence targeting of the 16S rRNA gene has been adopted for microbial communities associated with field-grown plants. To examine phylogenetic drifts according to read length and bioinformatic tools, original and chopped sequences (250–570 bp) covering the V1–V4 regions of 16S rRNA genes were compared using pyrosequence and Sanger reads of rice root microbiomes. The phylogenetic assignment at genus level depended on read length, especially in the genus  Diverse microorganisms live in and on plants. Microbial communities of field-grown plants have been surveyed by the use of 16S rRNA sequence-based methods in many studies. In most cases, clone libraries were constructed by PCR amplification of 16S rRNA gene segments , 13, 14.et al. unpublished). Pyrosequencing yields more than half a million sequences per run, and enables analysis of many more samples simultaneously at considerably lower cost; however, its limitations are short and variable read lengths, and a lower sequence quality than those of Sanger dideoxy sequencing (http://rdp.cme.msu.edu/) Classifier tool is accurate down to genus level  has now been used to analyze plant-associated bacterial communities  cultivar Nipponbare was grown in an experimental field at Tohoku University  in 2009, and bacterial DNA was extracted from the roots using the bacterial cell enrichment method , MID-518R (5′-CCATCTCATCCCTGCGTGTCTCCGA CTCAG_ barcode (MID)_ ttaccgcggctgctgg-3′), where nucleotide sequences shown in lowercase letters are universal sequences of bacterial 16S rRNA genes. MID-518R primer contains the sequences of the Titanium A adaptor, Key sequence (TCAG) and barcode sequences (MIDs), while Bac-27F primer contains the sequences of the Titanium B adaptor and Key sequence (TCAG). These PCR primers target the V1–V3 regions  were used to simulate the effect of read length on the taxonomic assignment of 16S rRNA gene reads. Regions corresponding to the first 250, 300, and 350 bases of the 518R primer were independently retrieved from the region between the 518R and 109F primers, and were designated S518R-250, -300, and -350 samples (http://pyro.cme.msu.edu/init/form.spr) for maximum edit distances of primer 518R=0 and of primer 109F (5′-ACGGGTGM-GTAACRCGT-3′)=2. Potentially chimeric sequences were removed with Chimera Slayer software  and 1525R (5′-AAGGAGGTGWTCCARCC-3′), and sequences were analyzed on a Type 3730xl DNA Analyzer  using the 27F primer. As for the Sanger reads, sequences that did not contain a perfect match with the 518R primer sequence were removed. The remaining sequences designated S665R-109F  was much lower in P518R-27F than in the other 454 samples, suggesting that the region between primers 518R and 27F was not suitable for detecting Firmicutes.Among the 454 samples analyzed using the RDP Multi-Classifier tool with a minimum support threshold of 80%, P518R-400, P518R-109F, P518R-350, and P518R-300 and P518R-250 showed almost identical phylogenetic compositions down to family level  On the o samples , which wystaceae . Interes samples , suggestd length . These rBradyrhizobium was extremely low at 250 bp (S518R-250 and P518R-250) as compared with other lengths of sequences . Sequences assigned as environmental samples  and unclassified sequences  were removed from the downloaded file. Remaining sequences were used as a BLASTN database. Taxonomic assignment for each simulated sample was conducted according to best-hit pairs in BLASTN analysis  against the in-house SILVA SSU Ref NR database. BLASTN results were filtered by e-value (≤1.0e-30), hit length coverage (≥90% of a query sequence) and similarity (≥90%). The taxonomic assignment by BLASTN analysis (Methylosinus even in 250-bp sequences (S518R-250 and P518R-250), no assignment to Bradyrhizobium was observed using 250-bp Sanger and 454 sequences  from the other two samples (17–20%) (Bradyrhizobium remained stable (28–33%). Three independent analyses of 454 samples showed how the perceived phylogenetic diversity can be easily influenced by experimental procedures, emphasizing the need for multiple independent analyses to reduce bias  . On the uce bias . It is iuce bias , DNA exttable 28–3%. Three


Salmonella enterica subsp. enterica serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 S. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H2S-negative S. Choleraesuis isolates and two H2S-positive isolates. This is the first report of H2S-negative S. Choleraesuis isolated from humans. The majority of H2S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the gyrA and parC genes and identified two new mutations in the parC gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H2S-negative and H2S-positive S. Choleraesuis isolates. PFGE revealed two groups, with all 19 H2S-negative S. Choleraesuis isolates belonging to Group I and H2S-positive isolates belonging to Group II. By MLST analysis, the H2S-negative isolates were all found to belong to ST68 and H2S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H2S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H2S-negative isolates also possessed a frame-shift mutation at position 760 of phsA gene compared with H2S-positive isolates, which may be responsible for the H2S-negative phenotype. Moreover, the 19 H2S-negative isolates have similar PFGE patterns and same mutation site in the phsA gene, these results indicated that these H2S-negative isolates may have been prevalent in China. These findings suggested that surveillance should be increased of H2S-negative S. Choleraesuis in China. Salmonella is a major health threat in both developing and developed countries, with an estimated 93.8 million cases and 155,000 deaths each year [Salmonella enterica subsp. enterica serovar Choleraesuis, which has adapted to swine, primarily causes septicemia, pneumonia and diarrhea [S. Choleraesuis was reported to be the second most common serovar causing human salmonellosis in Taiwan and the eleventh most common serovar in Thailand [S. Choleraesuis is not often isolated from humans [S. Choleraesuis infection of humans.Salmonellosis is recognized as an important public health problem causing human gastroenteritis and bacteremia throughout the world. The spread and infection of this pathogen is caused by the consumption of contaminated food or water in humans and animals . Nontyphach year . Salmonediarrhea . This padiarrhea –8. S. ChThailand . Apart fm humans . ContamiSalmonella and other intestinal bacteria, selective medium such as deoxycholate hydrogen lactose (DHL) or Salmonella-Shigella (SS) agar are the conventional methods. As a typical phenotype of Salmonella, the production of hydrogen sulfide (H2S) is detected through Triple Sugar Iron Agar (TSI). Salmonella has been considered to be typical H2S-producer, however many serovar of Salmonella, such as S. Choleraesuis, have been reported to be non-H2S-producer recently [To identify recently –16.S. Choleraesuis isolates identified during national surveillance of salmonellosis in China between 2010 and 2011. The majority of these isolates (90%) exhibited a H2S-negative phenotype. To our knowledge, human infections caused by H2S-negative S. Choleraesuis isolates have not previously been reported. The 21 S. Choleraesuis isolates were analyzed by antimicrobial susceptibility testing. We also performed pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis to evaluate the molecular characteristics and genetic relationships of these isolates [In this study, we examined 21 isolates –20.The study was approved and authorized for each location by the institutional ethics committees of the Academy of Military Medical Sciences of the Chinese People's Liberation Army, Beijing, China. The institutional review board of the Academy of Military Medical Sciences waived the need for written informed consent from the participants.Salmonella-like colonies were separated from 21 individual patients with diarrhea in China between 2010 and 2011. These presumptively Salmonella-like colonies cultured on SS agar  at 37°C for 24 h, and the colorless colonies indicated a H2S-negative phenotype. All of the colorless colonies were further identified using API 20E biochemical test kits  and were serotyped by serological testing  according to standard methods. To confirm H2S production, the colorless colonies were subcultured on TSI agar at 37°C for 24 h. All isolates (including two H2S-positive and ninteen H2S-negative isolates) were used for further analysis.Twenty-one presumptively S. Choleraesuis isolates was tested against 21 different antibiotics using broth microdilution in a 96-well microtiter plate  and the results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [Escherichia coli ATCC 25922 was used as a control strain. To clarify the molecular mechanism of fluoroquinolone resistance, the gyrA and parC genes were amplified by PCR using the primers described in The antimicrobial susceptibility of the 21 S. Choleraesuis isolates was carried out using the protocols described at http://mlst.warwick.ac.uk/mlst/dbs/Senterica. Total DNA of S. Choleraesuis was extracted using a TIANamp Bacteria DNA Kit  according to the manufacturer’s directions. The primers and PCR conditions used were the same as previously described [S. Choleraesuis was uploaded to the MLST database and determined the sequence type (ST). STs were analyzed for clonal relationship using the eBURST version 3 program (http://ebrust.mlst.net) [MLST of the 21 escribed . The PCRlst.net) .S. Choleraesuis isolates included in this study were analyzed by PFGE according to the standard methods outlined by PulseNet [XbaI restriction enzyme  at 37°C for 3 h. Then, electrophoresis of the digested DNA was performed using a CHEF Mapper PFGE system  using 1% SeaKem agarose in 0.5× Tris-borate-EDTA for 19 h. Macrorestriction patterns were compared using BioNumerics Fingerprinting software version 6.01 . The dendrogram was based on unweighted pair-group method by use of average linkages and pairwise Dice coefficients.All 21 PulseNet . The DNAhttp://crispr.u-psud.fr/Sever/) [Salmonella (http://www.pasteur.fr/recherche/genopole/PF8/crispr/CRISPRDB.html). No matching spacers or direct repeats were assigned according to appropriate spacer nomenclature [A previously described strategy was used in this analysis . We used/Sever/) . Spacersnclature . We usednclature .phs operon, containing the phsA, phsB, and phsC genes, is essential for the generation of H2S [S. Choleraesuis strain SC-B67 (NC_006905.1) and are shown in The n of H2S . PrimersS. Choleraesuis isolates from 21 individual patients, including six (29%) from Nanjing and fifteen (71%) from Shanghai. All the isolates were recovered from diarrhea patients. Phenotype analysis revealed that, there were nineteen H2S-negative and two H2S-positive S. Choleraesuis by H2S phenotype analysis , followed by ampicillin (74%), gentamicin (74%), ticarcillin (74%), trimethoprim-sulfamethoxazole (63%), tetracycline (58%), tobramycin (42%), and piperacillin (26%). Resistance was not detected to amikacin, aztreonam, cefazolin, cefepime, cefoperazone, cefoxitin, ceftazidime, ceftriaxone, imipenem, levofloxacin, or nitrofurantoin. Fifteen (79%) H2S-negative S. Choleraesuis isolates were multidrug resistant  (Of the 21  agents) .2S-negative S. Choleraesuis isolates were intermediate or resistant to norfloxacin, a fluoroquinolone. We detected mutations in the gyrA and parC genes resulting in fluoroquinolone resistance. Half of the isolates were only detected single-site mutation in the parC gene . Although these mutations are novel, further work is required to demonstrate whether these mutations contribute to resistance.Moreover, six (32%) of the HarC gene . In the isolates . HoweverS. Choleraesuis isolates were divided into two MLST STs, designated ST68 and ST145, by eBURST analysis. The two STs identified in this study both belonged to clonal complex 6 (CC6) and four isolates from Nanjing (SC1201–1204). The remaining two isolates from Nanjing (SC1205 and SC1206) were typed as ST145. These results suggested that ST68 may be the most prevalent ST in Shanghai and Nanjing.The 21 x 6 (CC6). ST145 wS. Choleraesuis isolates were subtyped into 16 unique XbaI PFGE patterns  and four from Nanjing (SC1201–1204) that shared approximately 96% similarity in their PFGE patterns. This finding indicated that H2S-negative S. Choleraesuis isolates belonging to ST68 have the potential for cross-region dissemination. In Group II, there were only two isolates, which belonging to ST145 were identified as H2S-positive, and shared approximately 96% similarity as well.The 21 patterns . The 19 All isolates belonging to ST68 were found to possess the same five spacers in CRISPR 1 . Two isophs operon of all 21 S. Choleraesuis isolates and compared it with that of S. Choleraesuis strain SC-B67. In all 19 H2S-negative isolates, a single base deletion was detected at position 760 in the phsA gene (2S. No mutations in the phsB and phsC genes were detected (data not shown). These results suggested that the H2S-negative phenotype may result of the frame-shift mutation at position 760 in the phsA gene .We analyzed the hsA gene . This de2S-negative Salmonella isolates identified from chicken, pork, seafood, and humans has been reported globally in recent years [2S-negative Salmonella, including S. Derby, S. Heidelberg, S. Rosenthal, S. Enteritidis, S. Indiana, S. Typhimurium, S. London, and S. Virchow [2S-negative S. Typhimurium and S. Infantis strains in retail chicken meat and found nonsense mutations at positions 1440 and 358, respectively, in the phsA gene [2S-negative S. Senftenberg isolates worldwide with a nonsense mutation at position 1621 of the phsA gene [2S-negative isolates among 21 S. Choleraesuis isolates detected in China during the period from 2010 to 2011. A frame-shift mutation at position 760 of the phsA gene was detected in all 19 H2S-negative S. Choleraesuis isolates, indicating that phsA gene maybe unstable and is likely responsible for the H2S-negative phenotype. It has been shown that the accumulation of thiosulfate (S2O32–), which was unable to be converted to H2S by H2S-negative S. enterica in the intestines, could promote S. Typhimurium survival and inflammation due to tetrathionate (S2O3–) respiration by reducing thiosulfate (S2O32–) consumption [2S-negative S. Choleraesuis may possess growth advantages when competing with other microbes in the lumen and may have stronger survival abilities compared with H2S-positive isolates. Furthermore, the incidence of infections caused by H2S-negative Salmonella isolates may have been greatly underestimated because of their atypical biochemical characteristics and the methods of detection employed. Taken together, the appearance of H2S-negative isolates may present an even greater challenge in the surveillance and control of Salmonella.The incidence of Hnt years –16. In ChsA gene . We prevhsA gene . Here, wsumption –30. Thus2S-negative S. Choleraesuis isolates analyzed in this study all belonged to ST68, which is a single locus variant of ST145, and shared some spacers with the isolates belonging to ST145 in the CRISPR 1 and 2 loci. These results indicated that the H2S-negative isolates may be derived from isolates belonging to ST145. The 19 H2S-negative isolates were also found to share similar PFGE profiles and possessed the same mutation in the phsA gene. These data provide evidence that the 19 H2S-negative S. Choleraesuis isolates are closely related genetically and may have developed into a new subgroup in Eastern China. Moreover, three H2S-negative isolates from Shanghai shared similar PFGE patterns with four H2S-negative isolates from Nanjing, with approximately 96% similarity in Group I. This suggested that H2S-negative S. Choleraesuis may disseminate across regions and may have been prevalent in Shanghai and Nanjing. Therefore, more attention should be paid to avoid further dissemination of H2S-negative S. Choleraesuis isolates in China.The 19 H2S-negative S. Choleraesuis isolates were multidrug resistant and most of the isolates exhibited high resistance to conventional antibiotics, such as chloramphenicol, ampicillin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. Among the H2S-positive S. Choleraesuis isolated from humans have been reported, the prevalence of multidrug resistance was 76% in Taiwan and nearly 90% in Thailand [2S-negative S. Choleraesuis isolates detected in this study displayed high rates of multidrug resistance as well. In Taiwan, most H2S-positive isolates (64%) exhibited resistance to the fluoroquinolone, norfloxacin [2S-negative isolates were found to possess intermediate or full resistance to norfloxacin. One mechanism of fluoroquinolone resistance was reported to be associated with the presence of multiple mutations in the gyrase genes (gyrA and gyrB) and the topoisomerase IV genes (parC and parE) [S. Choleraesuis isolates [gyrA and parC genes simultaneously, indicating that the instability of these genes may enhance resistance to norfloxacin. Moreover, cephalosporin-resistant isolates have frequently been detected in S. Choleraesuis and may therefore pose another threat to public health [2S was suppressed, bacteria could increased the sensitivity to a multitude of antibiotics [2S is cytoprotective against antibiotics. This phenomenon could explain our finding that all 19 H2S-negative S. Choleraesuis isolates exhibited susceptibility to cephalosporins.Fifteen (79%) of the HThailand ,32.Compafloxacin . In our nd parE) –36. It hisolates ,37–40. Ac health ,42. Howeibiotics . These r2S-negative S. Choleraesuis were detected in Eastern China. These isolates, which display a high prevalence of multidrug resistance, may pose a new threat to public health in the future. In response, we should strengthen surveillance for the appearance and spread of H2S-negative Salmonella isolates in China and throughout the world.In this study, a high proportion of HS1 Fig2S-positive S. Choleraesuis exhibited the black colony, while the H2S-negative S. Choleraesuis exhibited the colorless colony.H(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


To further exploit the excellent PSMA-targeting characteristics and in vivo performance of the PSMA-I&T platform, [111In]PSMA-I&T was evaluated as a complementary probe for radioguided surgery and SPECT imaging.The relevance of prostate-specific membrane antigen (PSMA) targeting in the clinical management of prostate cancer (PCa) is continually increasing, entailing the development of PSMA-targeted molecular probes. Recently, a first PSMA-targeted theranostic concept has been successfully implemented by [68Ga/177Lu]PSMA-I&T, [111In]PSMA-I&T showed unchangedly high PSMA-affinity and enhanced internalization into PSMA-expressing LNCaP PCa cells. Biodistribution studies in LNCaP xenograft-bearing mice (1 h p.i.) revealed slightly reduced background accumulation of [111In]PSMA-I&T compared to [177Lu]PSMA-I&T and identical tumor uptake of both compounds, leading to increased tumor/background ratios for [111In]PSMA-I&T. An exemplary patient with metastatic PCa underwent preoperative [68Ga]HBED-CC-PSMA PET/CT (1 h p.i.) and [111In]PSMA-I&T SPECT/CT (4 h p.i.), followed by prostatectomy and radioguided extended pelvic lymphadenectomy (24 h p.i.). In [111In]PSMA-I&T SPECT/CT, the previously identified PCa lesions ([68Ga]HBED-CC-PSMA PET/CT) showed high tracer accumulation and were also detectable using planar scintigraphy. The intraoperative use of a hand-held gamma probe allowed detection and resection of all [111In]PSMA-I&T-accumulating lesions. The presence of PSMA-positive tumor tissue in the resected specimens was confirmed histopathologically and via [111In]PSMA-I&T autoradiography.Compared to [111In]PSMA-I&T shows efficient PSMA targeting in vitro and in vivo, combined with low background accumulation. In an exemplary PCa patient, [111In]PSMA-I&T was successfully applied for preoperative SPECT/CT visualization and radioguided resection of PSMA-positive lesions, hinting towards a high value of [111In]PSMA-I&T as a complementary tool to [68Ga/177Lu]PSMA-I&T in the clinical management of prostate cancer.[ Modern clinical management of prostate cancer increasingly relies on exploiting the prostate-specific membrane antigen (PSMA) as a molecular target both for imaging and for treatment of prostate cancer (PCa). PSMA is abundantly expressed on the surface of prostate cancer cells and within the neovasculature of other solid tumors, with limited expression in most normal tissues, establishing the basis for selective targeting of PCa lesions with PSMA-targeted agents. Among the rapidly increasing number of high-affinity PSMA ligands, ranging from intact antibodies to low-molecular-weight compounds, urea-based inhibitors have been most extensively leveraged, with expanding clinical use .68Ga/177Lu]PSMA-I&T [68Ga/177Lu]PSMA-I&T, permitting high-contrast PET imaging of metastatic PCa with [68Ga]PSMA-I&T and, based on suitable uptake and retention characteristics, efficient treatment with its therapeutic analog [177Lu]PSMA-I&T PSMA-I&T  = 173, 245 keV) for the intended applications, PSMA-I&T was labeled with 111In using a standard protocol and was evaluated preclinically and in a first patient.The success and ease of implementation of this theranostic approach, relies, among other factors, on the ability of DOTAGA to form stable complexes with a broad variety of radiometals  and on t&T Table  2]. In . In 3+]P&T Table . Given tnatIn]PSMA-I&T shows unchangedly high PSMA affinity in a competitive binding assay (IC50), which equals that of its natLu-counterpart. Unexpectedly, however, internalization efficiency of [111In]PSMA-I&T into LNCaP prostate cancer cells was found to be markedly enhanced compared to [177Lu]PSMA-I&T . Particularly high tracer uptake in spleen and kidney in CB17 SCID mice has been consistently observed in the evaluation of a variety of PSMA-targeted radiopharmaceuticals in our lab and is mouse-strain specific.Besides high tumor uptake, 11InPSMA-111In]PSMA-I&T, especially in liver and intestines, are tracer specific. Due to its reduced lipophilicity compared to [68Ga]PSMA-I&T and [177Lu]PSMA-I&T (see Table 111In]PSMA-I&T shows improved tumor(t)-to-background ratios compared to [177Lu]PSMA-I&T at 1 h p.i., i.e., t/blood-, t/liver-, t/intestines-, and t/muscle-ratios of 34 ± 8, 32 ± 6, 53 ± 8, and 43 ± 6, respectively, versus 18 ± 9, 7 ± 3, 12 ± 3, and 14 ± 9 for [177Lu]PSMA-I&T.In contrast, the accelerated blood clearance and reduced background accumulation of 11InPSMA-111In]PSMA-I&T in comparison to [68Ga]PSMA-I&T and [177Lu]PSMA-I&T, an exemplary patient study was carried out to establish the potential of [111In]PSMA-I&T as an intraoperative probe for radioguided surgery and to investigate its suitability as a SPECT-imaging agent. The patient (51 years old), presenting with histologically confirmed metastasized prostate cancer , initially underwent [68Ga]HBED-CC PSMA PET/CT for pre-therapeutic staging as previously described PSMA-I&T, and preoperative planar scintigraphy as well as SPECT/CT were performed at 4 h p.i.  as well as in pelvic and retroperitoneal lymph node metastases (dotted arrows). Axial [111In]PSMA-I&T SPECT/CT images confirm the intense tracer accumulation, both in the primary tumor  correlating well with a H&E stained slide from histopathology (J), demonstrating the sensitive detection of PSMA-expressing tumor cells by [111In]PSMA-I&T.Ventral and dorsal views of whole body planar scintigraphy Fig. b show inmor Fig.  and in amor Fig.  and are ngs Fig. . During 111In]PSMA-I&T as a PSMA-targeted intraoperative probe has recently been further pursued [111In]PSMA-I&T radioguided surgery was shown to represent a valuable technique for the intraoperative detection of small subcentimeter metastatic lymph nodes and atypically located lesions.Based on these initial promising results, the concept of radioguided lymphadenectomy in early recurrent prostate cancer patients using PSMA-I&T showed promising in vivo characteristics as a PSMA-targeted SPECT imaging probe, including suitable whole-body clearance, predominant renal excretion, and efficient accumulation in PSMA-expressing tissues. Of course, in a direct comparison, [68Ga]HBED-CC PSMA PET/CT appears as the clearly superior imaging modality compared to [111In]PSMA-I&T SPECT/CT. However, this comparison is strongly biased in favor of [68Ga]HBED-CC PSMA PET/CT due to the inherent instrumental differences between PET and SPECT with respect to sensitivity and resolution. Furthermore, the comparably high gamma energy of 111In further challenges spatial resolution of [111In]PSMA-I&T SPECT/CT.Additionally, [68Ga]PSMA-I&T, [177Lu]PSMA-I&T, and [111In]PSMA-I&T as valuable tools for their respective dedicated application.However, the present preclinical and first clinical data underline the suitability of PSMA-I&T as a versatile labeling platform for a variety of PSMA-targeted applications in nuclear oncology and urology, ranging from PET and SPECT to endoradiotherapy and radioguided surgery, encouraging the ongoing further investigation of [


Human cancer cells may suppress the adaptive immune response by expressing PD-L1 and down-regulating T cell activity through PD-L1/PD-1 and PD-L1/B7.1 interactions. Disruption of PD-L1 signaling restores anti-tumor immunity, resulting in durable responses across multiple human tumor types. Here we describe the clinical activity and development of predictive biomarkers for MPDL3280A, a human monoclonal antibody with an engineered Fc-domain designed to optimize safety and efficacy, that targets PD-L1 and prevents binding to receptors PD-1 and B7.1.We evaluated a multitude of biomarkers from pretreatment tumor specimens collected during clinical study of MPDL3280A. MPDL3280A has been administered as a monotherapy in over 300 pts with locally advanced or metastatic solid tumors in a phase 1 study.To date, MPDL3280A has been well tolerated across multiple dose levels and no G3-5 pneumonitis has been reported. Responses were observed in multiple tumor types, including NSCLC, RCC, and melanoma, with ongoing responses seen in the majority of responders. We report the association between pretreatment immune-related markers and response to MPDL3280A.Overall, our assessment of tumor specimens not only has resulted in markers that may potentially identify response to MPDL3280A but also has furthered our understanding of the biologic activity of PD-L1 inhibition on treatment.


SMPD1) gene are associated with decreased catalytic activity of acid sphingomyelinase (ASM) and are the cause of the autosomal recessive lysosomal storage disorder Niemann-Pick disease (NPD) types A and B. Currently, >100 missense mutations in SMPD1 are listed in the Human Gene Mutation Database. However, not every sequence variation in SMPD1 is detrimental and gives rise to NPD. We have analysed several alleged SMPD1 missense mutations mentioned in a recent publication and found them to be common variants of SMPD1 that give rise to normal in vivo and in vitro ASM activity. .Loss-of-function mutations in the sphingomyelin phosphodiesterase 1 ( SMPD1) gene are associated with decreased catalytic activity of acid sphingomyelinase (ASM) and are the cause of the autosomal recessive lysosomal storage disorder Niemann-Pick disease types A and B (NPD). Currently, >100 missense mutations in SMPD1 are listed in the Human Gene Mutation Database. However, not every sequence variation in SMPD1 is detrimental and gives rise to NPD.Loss-of-function mutations in the sphingomyelin phosphodiesterase 1 -c.973C>G (p.P325A), c.1460C>T (p.A487V) and c.1522G>A -as well as a bipartite polymorphic site including the non-synonymous SNP c.107T>C , which is located adjacent to a polymorphic region composed of a variable number of hexanucleotide sequences. Two of these variants, p.P325A and p.A487V, were previously reported to constitute loss-of-function mutations of SMPD1 [SMPD1 genotypes with the respective in vivo ASM activities) and, second, cloned the respective cDNAs for transient transfection studies. Hereby, we were able to demonstrate that the carriers of c.1460C>T  displayed secreted (S-) and lysosomal (L-) ASM activities in the normal range [in vivo and in vitro [in vivo nor in vitro (In order to identify mutations in the 3C>G p.P35A, c.146in vitro . In contin vitro .SMPD1. These polymorphisms might increase the susceptibility for common diseases such as allergy [et al. [Our results strongly support the notion that p.V36A, p.A487V and p.G508R are frequent polymorphisms in  [et al.  are comm


The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates.  •Th1 genes and Th2 genes are associated with RNA pol II in the alternative lineage•T-bet acts to recruit Mediator and the SEC to activate Th1 genes and eRNAs•T-bet and NF-κB-dependent P-TEFb recruitment pathways converge at enhancers•P-TEFb inhibition silences T-bet target genes and abrogates uveitis in vivo Lineage-specifying transcription factors control T helper cell fate choice, but the mechanisms underlying this are unclear. Hertweck et al. reveal that Th1 genes undergo transcriptional initiation in the alternative Th2 lineage and that the Th1 master regulator T-bet functions to recruit Mediator and the super-elongation complex to activate transcriptional elongation. The differentiation of T helper cells into specialized effector lineages is a powerful model for understanding how master regulator transcription factors establish cell identity. Upon encounter with antigen, naive T cells can differentiate into one of several effector lineages . The parIfng. T-bet activates Ifng by binding at the gene itself and to multiple enhancer elements spanning a 146 kb region up and downstream , regions defined as super-enhancers are highly occupied by the co-activators Mediator, CBP, and p300, the BET protein Brd4, cohesin, and the Lsd1-NuRD complex . The higUsing a combination of primary human T cells and mouse models, we report here that Th1 genes undergo transcriptional initiation in Th1 and Th2 cells and that T-bet activates Th1 genes through recruitment of Mediator and the super elongation complex, with Brd4 instead recruited in a parallel pathway dependent on NF-κB.We hypothesized that profiling total RNA pol II occupancy by chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) in human Th1 and Th2 cells would provide insight into how T-bet functions to regulate gene expression. We first defined Th1 and Th2 genes as those expressed differentially across multiple samples, as done previously see . As expeWe sought to confirm transcriptional initiation of Th1 and Th2 genes in both lineages by measuring the initiation marker histone H3 trimethylated at lysine 4 (H3K4me3) in cells polarized for extended periods of time 28 days; . As we fcis-regulatory regions  test]; We next considered how T-bet function may be linked to lineage-specific transcriptional elongation and hypothesized that differential recruitment of the elongation factor P-TEFb may be involved. ChIP-seq for P-TEFb revealed increased recruitment in activated Th1 cells to genes with T-bet super-enhancers F. In conIn addition to binding at genes, we unexpectedly found that P-TEFb also bound extensively to intergenic sites, with 35% of sites located outside of genes B and 2C,The high-level of P-TEFb binding at Th1 and Th2 genes argues that the switch from transcriptional initiation to elongation is a particularly critical control point in the expression of these genes. To test this, we measured gene expression in in vitro differentiated mouse Th1 and Th2 cells in the presence of the P-TEFb inhibitor Flavopiridol or the BET inhibitor JQ1, which inhibits P-TEFb recruitment . We founWe next assessed whether the sensitivity of Th1 genes to elongation inhibition was related to super-enhancer function. We first identified the set of T-bet super-enhancers in mouse Th1 cells using replicate T-bet ChIP-seq data . Then, dAlthough JQ1 repressed a number of key Th1 genes E, the ef−/− mice cultured under Th1 conditions. We found that in the absence of T-bet, P-TEFb showed reduced binding to super-enhancers and to their associated genes  and T-beted genes A. Thus, To determine if T-bet was sufficient to recruit P-TEFb, we employed two murine EL4 cell lines, one that stably expresses T-bet and GFP and a control line expressing GFP alone . ChIP-seWe next explored how T-bet functioned in the recruitment of P-TEFb to super-enhancers and their associated genes. P-TEFb can be recruited to genes through NF-κB , the BETIfng, we found that T-bet induced binding of each protein to the promoter and to multiple distal regulatory elements that surround the gene RNAs. We found that the expression of T-bet target genes such as Ifng PCR. We found that the level of each eRNA tested was reduced in T-bet deficient cells, demonstrating that T-bet acts to induce eRNA transcription  model, in which infiltration of interferon-γ-producing CD4+ T cells into the retina can be induced by immunization with interphotoreceptor retinoid-binding protein (IRBP) . IRBP peHow T-bet regulates Th1 gene expression across the genome has been unclear. We show here that Th1 and Th2 genes undergo transcriptional initiation in a lineage-independent manner and that T-bet acts through extended regulatory regions (super-enhancers) to allow recruitment of Mediator and P-TEFb in the form of the SEC to activate Th1 gene expression. T-bet is necessary for P-TEFb recruitment, not just to gene promoters, but to super-enhancers themselves, where it functions to activate eRNA transcription. P-TEFb inhibition specifically downregulates Th1 genes and alleviates pathology in a Th1 cell-dependent uveitis model. T-bet is not required for RelA or Brd4 recruitment to most sites at Th1 genes. Instead, T-bet and NF-κB-dependent pathways converge at super-enhancers to allow P-TEFb recruitment. These data thus provide insight into the mechanisms of T-bet function during Th1 lineage-specification in human and mouse.Mediator is a large multi-subunit complex that integrates signals from multiple transcription factors to modulate transcription, chromatin modification, and looping between promoters and enhancers . It is pIfng expression downstream of the T cell receptor (TCR)  . We showpathways  and may How lineage-specifying factors function to promote differentiation toward a defined lineage, but also maintain the functional plasticity observed between effector subtypes has been a key unresolved issue. Epigenetic profiling has previously suggested that the establishment of bivalent chromatin at key lineage-specific transcription factors may be important . Our resWe identified extensive association of P-TEFb with enhancers. Brd4  and the Our study into the mechanisms through which T-bet directs Th1 lineage-specification suggests potential therapeutic avenues. BET inhibitors have previously been used to repress transcriptional elongation in immune cells and protect against inflammation in a number of models . AlthougHuman and mouse naive T cells were isolated and cultured under Th1 and Th2 polarizing conditions for 13 days, as described . For H3K−/− mice were purchased from Taconic. B10.RIII mice were obtained from GlaxoSmithKline. Mice were housed at the KCL Biological Service Unit (BSU) or at the UCL Institute of Ophthalmology BSU. Animal experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 .C57BL/6 mice were purchased from Charles River Laboratories International. T-bet−7 unless indicated. Super-enhancers were identified with the ROSE algorithm . Differentially expressed genes were identified by rank-sum test (pfp < 0.05). Murine Th1 and Th2 genes and genes repressed by Flavopiridol and/or JQ1 were identified by applying fold-change expression thresholds. The significance of differences in expression between gene sets was assessed with a K-S test.Poly-adenylated RNA was isolated with Oliogtex (QIAGEN). rRNA was depleted from total RNA with Ribo-Zero Gold (EpiCentre). Libraries were prepared using the Illumina Directional mRNA-Seq Sample Prep and the NEBNext Multiplex Small RNA Library Prep kits and then sequenced on an Illumina HiSeq 2500. RNA-seq reads were filtered for quality and to remove adapters, aligned to hg19 using TopHat2, and transcripts identified with Cufflinks v2.1.1 using default settings.161–180 (Cambridge Peptides) and monitored by fundoscopy on days 8–9 (B10.RIII mice were immunized subcutaneously with 300 μg IRBPdays 8–9 . Flavopidays 8–9 . Single Naive mouse CD4+ T cells were activated under Th1 polarizing conditions and transduced with pMY-Thy1.1-miR-30 retrovirus expressing shRNAs targeting firefly luciferase, Aff4, Med1, or Med17. After 3 days, Thy1.1+ cells were purified by magnetic cell sorting. On day 7, cells were either restimulated with 2 μg/ml anti-CD3/CD28 for 6 hr or left unstimulated.R.G.J. and G.M.L. initiated the research program. A.H., C.M.E., M.E., P.A., P.L., D.J.C., V.L.C., G.M.L., and R.G.J. designed experiments. A.H., C.M.E., M.E., J.C.H.L., K.O., I.J., A.K., D.J.C., and R.G.J. performed experiments and interpreted data. R.G.J. led the bioinformatics work, with some analyses performed by A.H., C.M.E., and J.A. R.G.J. oversaw the research. A.H. and R.G.J. wrote the paper, with input from all authors.