IL23R gene with IBD (Crohn's disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23RR381 and IL23RQ381 haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23RQ381 was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23RQ381 positive donors. Our study shows conclusively that IL23RQ381 is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD.Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the  The inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), have long been recognized as having a genetic element to disease susceptibility IL23R) gene  on chromosome 1p31 confers the highest odds-ratio (OR) for disease development/lowest OR for disease protection Several genes in the Th17 pathway have been linked with IBD susceptibility, including IL23R, TNFSF15, STAT3, IL12B, CCR6 and JAK2 IL23R is most highly expressed on activated T cells, particularly of the Th17 subtype, natural killer (NK) cells and at lower levels on monocytes, macrophages and dendritic cells IL23R pairs with IL12RB1 to confer IL-23 responsiveness on cells expressing both receptor subunits As well as functioning as a mediator of cytokine production in Th17 pathway cells, several studies also suggest that IL23R is a key player in the proliferation and survival of Th17 cells Q381 could potentially influence the response of the IL23R-expressing cells such as Th17 cells in the host. Indeed, a recent study by Di Meglio and colleagues Q381 exerts its protective effects through attenuation of IL-23 induced Th17 function.Because of its role in T cell biology and the compelling genetic evidence, we hypothesized that IL23RR381 and IL23RQ381 haplotypes.We investigated this possibility using primary T cells from healthy donors carrying IL23RThe R381Q polymorphism is located between the transmembrane domain and the putative JAK2 binding site in the cytoplasmic portion of IL23R protein, and is absolutely conserved across different species . By virtR381 or IL23RQ381. IL23R positive cells were detected using proprietary monoclonal antibody against human IL23R , revealed significantly diminished population of IL23R positive T cell from IL23RQ381 positive donors compared to their IL23RR381 counterparts  from the same donors. IL23R expression is known to be activation dependent R381 T cells, while the IL23RQ381 T cells had slightly, but not significantly decreased number of IL23R positive cells  and IL23RQ381 (n = 13) positive donors showed that IL23RQ381 donors have decreased IL-23–dependent IL-17 and IL-22 production. Serum IL-22 levels were determined using enzyme-linked immunosorbent assay (ELISA), and we again observed a slight, but not significant, trend toward decreased IL-22 production in IL23RQ381 donors compared to IL23RR381 donors, in agreement with previously published data R381 and IL23RQ381 positive donor groups  and IL23RQ381 (n = 19) positive donors demonstrated that the cells from L23RQ381 positive donors had impaired IL-23-mediated IL-17A production and STAT3 phosphorylation.In the past several years, GWAS have lead to major breakthroughs in our understanding of the genetics of CD. However, there have been relatively few published studies functionally validating disease-associated variants so far Q381 to activate STAT proteins due to impaired association of JAK2 proteins with the cytoplasmic tail of the receptor. Arginine 381 is located near the JAK2 putative binding site . We observed decreases in cytokine levels in IL23RQ381 positive donors compared to IL23RR381 donors in peripheral blood. However, the difference was not significant, potentially because of the small number of available samples.One of the major advantages of our study was the availability of healthy donors willing to donate blood repeatedly for our primary T cells studies. It should be noted that, while we used T cells in our analysis, the protective effect of IL23RFor future studies it will be important to analyze a much larger cohort of donors. In addition, it will be interesting to conduct deep sequencing studies to identify additional genetic variants harbored in IL23R. Indeed, recent study by Monozawa and colleagues NOD2, ATG16L1) as well as environmental influences  on the risk of CD development.It is important to note that despite the R381 risk variant having an allelic frequency of greater than 90% in Caucasian populations, the prevalence of CD in North American populations is only approximately 0.2% IL23R is a key player in the proliferation and survival of Th17 cells, which are critical for host defense against bacterial, fungal and viral infections at mucosal surfaces Q381 is a hypomorphic IL23R allele that results in a decreased population of IL-23 responsive cells, leading to diminished IL-23-induced STAT3 phosphorylation. This provides an explanation for the protective role of R381Q in CD and other autoimmune disorders  according to the manufacturer's instructions. Human IL-17A levels were measured in supernatants from PBMCs isolated from genotype specific healthy donors and stimulated with anti-CD3 (2.5 ug/ml) and anti-CD28 (1 ug/ml) antibodies+/−IL-23 (5 ng/ml) for 2 days. Supernatants were harvested and analyzed using hIL-17A ELISA (eBioscience) according to the manufacturer's instructions.IL23R coding sequence was PCR amplified using following primers: forward ACAAGTTTGTACAAAAAAGCAGGCTTCATGACCAATCAGGTCACTATT - 3′5′ GG . Reverse ACCACTTTGTACAAGAAAGCTGGGTCCTACTTTTCCAAGAGTGA - 3′5′ – GGGG . PCR product was cloned into the pDONR221 donor vector . Construct encoding IL23RR381 was subsequently transferred into a gateway adapted pMSCVpuro plasmid using LR Clonase, and final destination construct was sequence verified.Gateway recombination cloning technology (Invitrogen) was used to create all constructs. Full-length wild-type R381 cDNA, cloned in the pMSCVpuro retroviral expression vector, was introduced into the same hIL-12RB1 containing BaF3 subclones by standard retroviral transduction. 293T cells in combination with the retroviral packaging vector pCL-Eco (Imgenex) were used as a packaging system. Twenty-four hours after transduction, BaF3 cells were put in 1 µg/ml puromycin (Clontech) medium to select transduced cells. Relative IL23R and IL12RB1 mRNA abundances were verified by qPCR.BaF3 cells To detect IL23R cell surface expression BaF3 cells were stained with biotinylated IL23R antibody 20G3.4 in combination with streptavidin-PE (eBioscience). Isotype control antibodies were used as negative controls (BD biosciences).Q381 heterozygous (GA) and 1 homozygous (AA).Blood samples were obtained from healthy donors after written informed consent was provided. Ethical approval for the use of this material was obtained from the Western Institutional Review Board. Genomic DNA was isolated from 138 healthy donors, and the R381Q variant (rs11209026) was genotyped using Applied Biosystems TaqMan SNP genotyping assay (assay ID: C_1272298_10). Eighteen donors were identified as being IL23R+ T cells were sorted from whole blood of IL23RR381 and IL23RQ381 positive donors using a human whole blood CD4 selection kit  according to the manufacturer's instructions (purity of the CD4+ cells after enrichment was >95%). Cells were seeded at 5×105 cells/ml and stimulated with a feeder mixture containing 1×106/ml irradiated  allogenic PBMC and 1×105/ml irradiated  JY cells, 1 µg/ml phytohemagglutinin (Sigma), and 200 IU/ml recombinant human IL-2 (Roche). Cells were cultured in Yssel's medium (Gemini Bio-Products) supplemented with 1% human serum. T cells were restimulated with the feeder mixture every 2 weeks. Cell surface expression of IL23R was analyzed by flow cytometry 6 days after stimulation.T cell lines were generated as previously described R381 and IL23RQ381 positive donors using UNI-SEP  pre-filled tubes containing a solution of 5.6% polysucrose and 9.6% sodium metrizoate . Cells were stimulated with anti-CD3 (2.5 ug/ml) and anti-CD28 (1 ug/ml) antibodies for 72 hours. IL23R cell surface cell surface expression was analyzed by flow cytometry using biotinylated IL23R antibody in combination with streptavidin-PE (eBioscience).PBMCs were isolated from whole blood of IL23R5 cells per well. IL-23 was added to a final concentration of 100 nM and a 1∶4 dilution series was established. After 72 h of stimulation cells were analyzed by the CellTiter-Glow Luminescent Cell Viability Assay according to the instructions of the manufacturer (Promega Corp). All experiments were performed in triplicate wells.T cells were plated in 96-well plates in triplicates at 5×105 cells per well. IL-23 was added to a final concentration of 100 nM and a 1∶2 dilution series was established. After 72 h of stimulation cells were analyzed by a 16-h pulse with 1 µCi/well [3H] thymidine (Amersham Biosciences) followed by harvesting using a 96-well plate harvester and counting by liquid scintillation. All experiments were performed in triplicate wells.T cells were plated in 96-well plates in triplicates at 5×10All data were collected on FacsCalibur and LSR II instruments (BD Biosciences) and analyzed using FlowJo software (Tree Star). A summary of all antibodies used in this study is provided in T cells were stained with biotinylated IL23R antibody 20G3.4 in combination with streptavidin-PE (eBioscience). Isotype control antibodies were used as negative controls (BD biosciences).Flow cytometric analyses of STAT phosphorylation were performed as previously described Whole blood was stimulated with 10 ng/ml of IL-23 or 10 ng/ml of rhIL-6 and incubated at 37°C for 15 min, fixed and lysed to halt signaling and lyse red blood cells. Cells were permeabilized and stained with phospho-STAT3 specific antibodies to detect activated STAT3.Intracellular staining was done as previously described R381 and IL23RQ381 positive donors using RNeasy Micro kit (Qiagen). High-Capacity cDNA Archive kit (Applied Biosystems) was used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7900 sequence detector (Applied Biosystems) with Applied Biosystems predesigned TaqMan Gene Expression Assays. The following probes were used: RORC (Hs01076112) and IL23R (Hs00332759). For each sample, mRNA abundance was normalized to the amount of GAPDH or RPL19 transcripts.Total RNA was extracted from freshly isolated PBMCs of IL23Rhttp://www.ebi.ac.uk/clustalw/).IL23R protein sequences from all the species available in Genbank were aligned with Clustal W in FASTA format Click here for additional data file.Figure S2Q381 positive T cell have comparable cell viability and proliferation rates to IL23RR381 cells. The mean percent (n = 4) of representative donors (A) cell viability and (B) proliferation rates after stimulation with IL-23 for 72 h. Data are representative examples of at least three independent experiments.Untransformed polyclonal IL23R(TIF)Click here for additional data file.Figure S3Q381 and IL23RR381 positive donors. ICS of cytokine production by PBMCs stimulated with anti-CD3/ CD28 dynabeads (A) representative donors for IL-22 and IL-17 and (B) IL-10 and INF-g (C) four donors per group is shown. Data are representative examples of at least three independent experiments. The Mann-Whitney test was used to calculate the p value.Cytokine levels are comparable in IL23R(TIF)Click here for additional data file.Table S1List of anti-human antibodies used in this study.(DOCX)Click here for additional data file.


Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood.in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays.In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer. Breast cancer is the most prevalent form of cancer in women worldwide accounting for 23% of the total cancer cases and ranks second overall . It is the second most common cancer affecting women in India Keratins, the largest family of intermediate filament proteins are epithelia predominant in their expression An important feature of keratins is that their expression is regulated by tissue type and also during differentiation The keratin pair of K8 and K18 is the first pair to be expressed in embryogenesis, and expression of this pair is restricted later to simple  There are differing reports about the role of K8/18 pair in breast cancer progression. K8/18 when expressed along with vimentin have been associated with drug resistance, invasion and metastasis in breast cell carcinomas and melanomas in-vitro invasion and metastasis in-vivo. Down-regulation of K8 in non-transformed cell line did not result in any phenotypic changes but in a transformed less invasive cell line down-regulation of K8 resulted in increase in anchorage independence, significant increase in motility and invasion in-vitro. These results indicate that K8 may modulate motility and invasion in breast cancer progression.In this communication we have made an attempt to decipher the role of K8/18 in neoplastic progression of mixed epithelia like breast. We have over-expressed K8 in an invasive breast cancer cell line (MDA MB 435) and down-regulated K8 expression in a non-transformed (MCF10A) and transformed less invasive breast cancer cell line (MDA MB 468). Our results show that over-expression of K8 in an invasive breast cancer cell line resulted in significant decrease in motility, All the protocols for animal studies were reviewed and approved by Institutional Animal Ethics Committee (IAEC) of ACTREC . IAEC is constituted under the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.2 atmosphere.The MDA MB 435 breast cancer cell line, derived from the pleural effusion of a 51-years old woman with breast carcinoma K8 gene cloned in pCDNA3 (Invitrogen) was used for over-expression of K8. The shRNA to K8 gene - shRNA 8.2 (previously described) To generate stable cell lines constitutively expressing K8, MDA MB 435 cells (P-54) were transfected using lipofectamine Plus (Invitrogen) according to the manufacture’s protocol with 2 µg of either the vector pCDNA3 alone or K8 expression vector pCDNA3-K8. The transfectants were plated in medium containing G418,  at a concentration of 1200 µg/ml. To determine whether K8 loss results in increased neoplastic transformation, MDA MB 468 (P-15) and MCF10A (P-9) cells were transfected using Superfect (Qiagen) and ICAfectin 441(Eurogenetic) respectively with 2 µg of either vector pTU6.PURO alone or K8 shRNA (shRNA 8.2) cloned in pTU6 puro vector. The shRNA 8.2 was previously designed 6 cells were fixed in 1% paraformaldehyde for 15 minutes at 4°C. The cells were then treated with FACS buffer  for 5 minutes. The cells further incubated with anti- K8 (M20 clone) monoclonal antibody for 45 minutes at 4°C. After incubation with primary antibody, cells were washed three times with FACS buffer. The cells were then incubated with Alexa-Fluor-488-conjugated anti-mouse-IgG secondary antibody (Molecular Probes) for 45 minutes at 4°C. Cells were then analysed with FACS Calibur  flow cytometer. The mean fluorescence intensity was measured in arbitrary units using Msiy Cell Quest software.The cells were grown for 48 hours and were harvested by trypsinization. For the assay, 1×10The following antibodies were used:K8 , β-actin  all from Sigma-Aldrich, USA, E-Cadherin , from BD Transduction Lab, β-4 integrin  from Santa Cruz Biotechnology, K7  from Thermo Scientific, Pierce UK), secondary antibody HRP-conjugated anti-mouse, anti-rabbit  For immunofluorescence experiments, the vimentin antibody (clone 13.2) Sigma Aldrich, USA and the keratin antibodies listed above were used at a dilution of 1∶200. The secondary antibody, Fluorosceinisothiocyanate (FITC)-conjugated sheep anti–mouse IgG , was used at a dilution of 1∶200. For flow cytometry secondary antibody Alexa flour 488 anti-mouse-IgG secondary antibody (A1101 Molecular Probes) was used.The keratins were extracted in high salt extraction buffer and processed as described previously 6 random primers. Real time PCR analysis was performed on 10 ng of cDNA using gene specific primers for K8 , for K18  SYBR green based real-time quantitative PCR was performed on the ABI 7900HT Fast Real Time PCR System . The relative gene expression was quantified by comparative Ct method using GAPDH  as house keeping control.Real time PCR analysis was carried out to determine the K8 and K18 levels for MDA MB 435 clones. A total of 2 µg of RNA was reverse transcribed using pdN5′-CGTTGGCTACCCGTGATATT-3′ and Reverse primer 5′-AAGAAGGCGATAGAAGGCGA-3′), and for K8 knockdown clones, real time PCR was performed for puromycin resistant gene (Forward-5′ CGCAGCAACAGATGGAAGGC-3′ Reverse primer: 5′-CCGCTCGTAGAAGGGGAGGT-3′). GAPDH primer recognizing genomic GAPDH gene (Forward primer: 5′ AGGGTCTACATGGCAACTG-3′ and Reverse primer: 5′- CGACCACTTTGTCAAGCTCA-3′) was used as reference to determine the copy number.Plasmid copy number of all the clones was determined by real time PCR analysis on the genomic DNA isolated from the clones and parental cells. To determine the plasmid copy number of K8 up-regulated clones, real time PCR was performed for neomycin resistant gene (Forward primer: 4HCO3:Acetonitrile (1∶1) for 15 minutes and then with 100% Acetonitrile. The gel pieces were then vacuum dried and further incubated with 10 mM Dithiotreitol at 56°C for reduction and with 55 mM Iodoacetamide in dark at room temperature for alkylation. The gel pieces were then washed with 1∶1 ratio of NH4HCO3: Acetonitrile. The final wash with 100% Acetonitrile was given to dehydrate the gel pieces were vacuum dried followed by an overnight digestion with 10 ng/µl trypsin in 25 mM NH4HCO3 After digestion, the peptides were extracted using 50% Acetonitrile and 5% TFA (Tri fluoro acetic acid) solution. The tryptic protein digests were reconstituted using 10% Acetonitrile and 0.1% TFA solvent before subjecting to mass spectrometry analysis. A reconstituted sample and Matrix α-CyanoHydroxyCinnamic acid (CHCA) (1∶1) were mixed properly and were loaded onto the ground steel MALDI plate and subjected to mass spectrometry (MS) analysis on Ultraflex-II MALDI TOF-TOF  machine.Keratins extracted using high salt buffer were run on SDS-PAGE. The bands from Coomassie stained gels were excised for the mass spectrometry analysis. Briefly, for the analysis, the gel pieces were washed with water and after thoroughly rinsing, the gel pieces were gradually dehydrated using 50 mM NHFor immunofluorescence cells were grown on coverslips for 48 hours and treated with 0.03% Triton X-100 in chilled methanol for 90 seconds and fixed in chilled methanol for 10 minutes at −20°C. The fixed cells were washed in PBS twice and incubated with respective primary antibodies at room temperature for 1 hour. The cells were further washed with PBS four times each for 10 minutes and incubated with the secondary antibodies at room temperature for 1 hour. Confocal images were obtained with a LSM 510 Meta Carl Zeiss Confocal system with an Argon 488 nm laser. All images were obtained using an Axio Observer Z.1 microscope numerical aperture (NA 1.4) at a magnification of 63X with 2X optical zoom.Cells were stained with Nile red for lipid droplets (marker of cell differentiation). Briefly, the stock solution of Nile red 1 mg/ml in acetone was diluted in PBS (1∶1000). The fixed cells  were incubated with diluted Nile red for 5 minutes at room temperature, rinsed with PBS and observed for the presence of lipid droplets by confocal microscopy. Confocal images were obtained with a LSM 510 Meta Carl Zeiss Confocal system with Helium/Neon 543 nm lasers. All images were obtained using an Axio Observer Z.1 microscope numerical aperture (NA 1.4) at a magnification of 63X with 2X optical zoom.y-axis) against time (on x-axis).Cells were seeded, in triplicate in a 96-well microtiter plate in 100 µl complete medium. Proliferation was studied every 24 hours up to a period of 4 days using MTT assay as described previously Cells were tested for anchorage-independent growth using soft agar colony-forming assay as described previously 2. Cells were observed by time lapse microscopy and images were taken every 10 minutes for 20 hours using an Axio CamMRm camera with a 103 phase 1 objective. Migration was measured using Axiovision software version 4.5 (Zeiss).The stably transfected cells were tested for change in motility by scratch wound as well as transwell assay. For scratch wound assay the cells were grown in 35 mm plates to 95% confluency. Cells were incubated with fresh medium, containing 10 µg/ml mitomycin-C for 3 hours. After incubation, medium was discarded and wounds were scratched with the help of sterile 2 µl pipette tip. The cells were fed with fresh medium and observed under an Axiovert 200 M Inverted Carl Zeiss microscope fitted with a stage maintained at 37°C and 5% CO5 cells were seeded into the top chamber with serum-free medium containing 0.1% BSA and medium containing 10% serum was added to the lower chamber of the Boyden chamber . The cells were incubated for 16 hours. Motility of cells to the underside of the membrane was detected by wiping the upper side with cotton swab and staining the underside cells with Mayer's Hematoxylin and Eosin (H&E) solution. The cells were counted under microscope in five random fields after staining. The assay was repeated thrice with 3 replicates each time.For assessing motility by transwell assay 2×105 cells were seeded into the top chamber with serum-free medium containing 0.1% BSA and medium containing 10% serum was added to the lower chamber of the Boyden chamber coated with 40 µl (1 µg/µl stock) Matrigel . Cells that invaded through the matrix to the bottom of the filter were then fixed and stained with H&E solution. After staining, the cells were counted under microscope in five random fields. The assay was repeated thrice with 3 replicates each time.Matrigel invasion assay was performed to check the invasive potential of the stably transfected clones. For the assay 2×106 cells were injected in the mammary fat pad of five SCID mice. Tumor volume (mm3) was calculated by the formula (1/2 × width × width × length) as previously reported To determine the tumorigenic potential 1×10t-test statistical analysis p<0.05 was considered significant.Two groups of data were compared by performing a . MDA MB 435 cells showed high expression of vimentin, while it was weak or undetectable in MDA MB 468 and MCF10A cells  or K8 expression construct (K8- pCDNA3). Three stable clones expressing K8  and empty vector transfected clone (K8Vc) were obtained. The clones K8C1 and C2 along with the vector control K8Vc were selected for further analysis . Up-reguTo determine whether loss of K8 leads to an increase in transformation in the MCF10A or MDA MB 468 cells, a previously described shRNA construct to K8, shRNA8.2 A differentiated mammary gland exhibits a specific secretory function of producing milk. Human milk mainly is constituted of fat globules containing triglycerides. Triglycerides are neutral lipids that can be stained by Nile red In order to assess the effects of K8 expression on tumorigenesis, soft agar colony forming assays were performed. The MDA MB 435 K8 over-expressing clones (K8C1 and C2) did not show any significant change in the size and the number of the colonies formed in the soft agar as compared to the vector transfected clone (K8Vc) . The K8 To determine if an alteration in K8 levels led to changes in proliferation, MTT assays were performed in the K8 over-expressing or K8 knockdown clones. The K8 over-expressed MDA MB 435 clones  showed significantly decreased growth as compared to the vector control clone (K8Vc)  . The MDAPreviously published results from our laboratory and others have suggested that alterations in K8 expression lead to changes in motility Our previous results have demonstrated that changes in K8 expression lead to changes in metastatic progression Changes in cell adhesion molecule like E-cadherin have been reported earlier in breast cancer Previous reports from our laboratory showed the involvement of β4 integrin mediated pathway on K8 down-regulation in human oral SCC derived cell lines in-vivo, MDA MB 435 derived K8 over-expressing clones of (K8C1 and C2) and the vector control clone (K8Vc) were injected in mammary fat pad of the SCID mice. Mice injected with K8 over-expressing clones demonstrated significantly smaller tumors as compared to the vector control clone  MCF10A, MDA MB 468 and MDA MB 435 cells using mAb to K8, K18 and vimentin respectively. (B) Western blot analysis of K8 and K18 in MCF10A, MDA MB 468 and MDA MB 435 cells using mAb to K8 and K18 respectively. β-actin was taken as loading control. Histogram showing relative intensity of K8 and K18 of the cell lines MCF10A, MDA MB 468 and MDA MB 435 (Right hand side). The fold change was calculated using Image J software. To determine the fold intensity, Intensity of the band was normalized using β-actin as internal control. Relative intensity was measured by considering band intensity of MCF10A for K8 and K18 as 1. MDAMB 468 showed 2 fold less K8 and K18 as compared to MCF10A while in MDA MB 435 K8 expression was negligible and K18 expression was 5 fold lesser than MCF10A. Note: Decrease in K8, K18 expression from MCF10A to MDA MB 435 cells.Analysis of K8, K18 and vimentin in MCF10A, MDA MB 468 and MDA MB 435 cell lines. Representative confocal images of K8, K18 and vimentin filaments formed in (TIF)Click here for additional data file.Figure S2(A) MCF10A, MDA MB 468 and MDA MB 435. (B) Histogram showing number of colonies formed in soft agar. (C) Histogram showing volume of colonies formed in soft agar. Size of the colonies was determined using Axiovision software (*p<0.05 by student’s t-test). Results are mean of ± SE of three independent experiments performed. Note: Increase in number and volume of soft agar colonies formed from non-transformed MCF10A to invasive MDA MB 435 cell lines.Analysis of soft agar colony forming potential in MCF10A, MDAMB 468 and MDA MB 435 cell lines. Representative phase contrast images (10X) of colonies formed in soft agar per plate by (TIF)Click here for additional data file.Figure S3(A) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing the mean intensity of MDA MB 435 K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (right hand side). (B) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing the mean intensity of MDA MB 468 K8 down-regulated  and vector control (Vc) clones (right hand side). (C) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clones. Histogram showing the mean intensity MCF10A K8 down-regulated  and vector control (MVc) clones (right hand side). Scale bars: 10 µm. All the scanning conditions of gain offset and laser percentage were kept same and applied for all images of vector and their respective clones with secondary control as threshold. The mean fluorescence intensity (± SE) of lipid droplets was calculated per cell by measuring fluorescence intensity of 20 cells of each experiment .This was repeated thrice Results are mean of ± SE of three independent experiments performed. Note: No change in lipid droplet staining intensity in any of the clones on K8 up−/down-regulation.Analysis of differentiation status of K8 up−/down-regulated clones by lipid droplets staining using Nile red. Representative confocal images of lipid droplets staining (TIF)Click here for additional data file.Figure S4. Note: Keratin 8 was observed to be down-regulated in the K8 knockdown clones as compared to vector control.High salt Keratin extraction. The keratin profile of MDA MB 468 K8 down-regulated (ShC1 and C2) and vector control (Vc) clones after high-salt extraction. The arrow indicates position of K8 band on the gel at molecular weight (M.W. ∼52 kDa). The numbers indicated on the left hand side indicates the gel pieces taken for the mass spectrometry analysis(TIF)Click here for additional data file.Figure S5(A) Histogram showing % protein expression (± S.E.) for three independent experiments of K8 in MCF10A K8 down-regulated  and vector control (MVc) clones.(B) Histogram showing % protein expression (± S.E.) for three independent experiments of K18 in MCF10A K8 down-regulated  and vector control (MVc) clones. The percentage of protein expression was determined by Image J software. The intensity of the K8 or K18 expression was normalized with β-actin. Vector control clone (Mvc) intensity was considered as 100% expression. Note: ∼60% down-regulation in K18 levels on K8 down-regulation.MCF10A K8/18 down-regulation. (TIF)Click here for additional data file.Figure S6(A) K8 up-regulated MDA MB 435 clones (K8C1 and C2) as compared to parental MDA MB 435 and vector control clone (K8Vc) (B) K8-down-regulated MDA MB 468 clones  as compared to parental MDA MB 468 and vector control clone (Vc). (C) K8 down-regulated MCF10A clones  as compared to parental MCf10A and vector control clone (Mvc) as analysed by flow cytometry.Analysis of K8 expression by flow cytometry in K8 up-regulated clones of MDA MB 435, K8 down-regulated clones of MDA MB 468 and MCF10A. Histograms showing mean fluorescence intensity of K8 (± S.E.) for three independent experiments (lower panel); in (TIF)Click here for additional data file.Figure S7(A) K8 gene in MDAMB 435 K8 over-expressed  and vector control (K8Vc) clones and (B) K18 gene in K8 over-expressed MDA MB 435 clones (K8C1 and C2) as compared to vector control clone (K8Vc) using GAPDH as internal control. Results are mean of ± SE of three independent experiments performed. Note: No significant difference in K18 levels on K8 up-regulation.Real time PCR analysis of K8 and K18 expression in K8 up-regulated MDA MB 435 clones. Real time PCR analysis of genes encoding (TIF)Click here for additional data file.Table S1Details of the bands with keratin identity.(DOCX)Click here for additional data file.


Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble  and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism  Yersinia pestis is a deadly disease that wiped out one-third of Europe's population in the 14th century. The organism is listed by the CDC as Tier-1 biothreat agent, and currently, there is no FDA-approved vaccine against this pathogen. Stockpiling of an efficacious plague vaccine that could protect people against a potential bioterror attack has been a national priority. The current vaccines based on the capsular antigen (F1) and the low calcium response V antigen, are promising against both bubonic and pneumonic plague. However, the polymeric nature of F1 with its propensity to aggregate affects vaccine efficacy and generates varied immune responses in humans. We have addressed a series of concerns and generated mutants of F1 and V, which are completely soluble and produced in high yields. We then engineered the vaccine into a novel delivery platform using the bacteriophage T4 nanoparticle. The nanoparticle vaccines induced robust immunogenicity and provided 100% protection to mice and rats against pneumonic plague. These highly efficacious new generation plague vaccines are easily manufactured, and the potent T4 platform which can simultaneously incorporate antigens from other biothreat or emerging infectious agents provides a convenient way for mass vaccination of humans against multiple pathogens.Plague caused by  Yersinia pestis, the etiologic agent of plague, is a Gram-negative bacterium transmitted from rodents to humans via fleas Y. pestis, leading to near 100% death of infected individuals within 3–6 days Y. pestis poses one of the greatest threats for deliberate use as a biological weapon http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm302220.htm), prophylactic vaccination is one of the most effective means to reduce the risk of plague.Plague, also known as Black Death, is one of the deadliest infectious diseases known to mankind. Y. pestis strain, KIM/D27  vaccine was in use in the United States, and a live attenuated plague vaccine (EV76) is still in use in the states of former Soviet Union Y. pestis virulence factors, the capsular protein , into the host cytosol  assemble into trimers at the quasi three-fold axes, clamping to adjacent capsomers and forming a reinforced cage around the shell  [34]. Thcapsomer . Hoc binH1 and TH2 responses. Both the soluble and T4 decorated F1mut-V provided 100% protection to mice and rats against intranasal challenge with high doses of Y. pestis CO92. Inclusion of YscF showed a slight enhancement in the potency of F1-V plague vaccine, whereas replacement of V with V10 mutant, which lacks the putative immunosuppressive sequence, did not significantly alter vaccine efficacy. These results provided new insights into plague vaccine design and produced next generation plague vaccine candidates by overcoming some of the concerns associated with the current subunit vaccines.Here, we describe two basic approaches to generate next generation plague vaccines, structure-based immunogen design and T4 nanoparticle delivery . We desi2-terminal β-strand of the “n+1” subunit locks in, resulting in a bridge connecting adjacent subunits (inter-molecular complementation)  . Stringi E. coli  exposes 2-terminal β-strand of F1 to the COOH-terminus should reorient the β-strand such that it fills its own cleft  by deleting the NH2-terminal donor strand [amino acid (aa) residues 1–14] and fusing it to the COOH-terminus with a short (Serine-Alanine) linker in between  was produced by fusing F1mut2 to the NH between , and itsive F1-V .Y. pestis V antigen has been reported to induce interleukin (IL)-10 and suppress the production of pro-inflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which could lead to lowering of innate immunity in vaccinated individuals E. coli, which was also highly soluble and could be purified as a monomer  was 663, which meant that ∼76% of the Soc binding sites were occupied, and its apparent binding affinity (Kd) was 292 nM, which was ∼4-fold lower than that of Soc binding (Kd = 75 nM) The 66 kDa F1mut-V-Soc bound to T4 even at a relatively low 1∶1 ratio of F1mut-V-Soc molecules to Soc binding sites   were immunized according to the scheme shown in 50 of Y. pestis CO92 [1 LD50 = 100 colony forming units (CFU) in Balb/c mice], one of the most lethal strains, showed that all the control mice died by day 3. However, the mice immunized with native V immunogen showed 83% survival (two of twelve mice died), whereas the mice immunized with F1-V, F1mut-V, or F1-V plus YscF were 100% protected   . All of 50 of Y. pestis CO92; all the control animals died by day 4. Upon re-challenge on day 48 post-first challenge with 9,800 LD50  and cellular (TH1), is probably essential for protection against Y. pestis infection H1 profile involves induction of antibodies belonging to IgG2a subclass whereas the TH2 profile primarily involves the induction of IgG1 subclass. To determine the specificity of antibodies induced by soluble vs T4 displayed antigens, the subclass IgG titers were determined by ELISA (H2 response) (H1 response)   whereas esponse) . These rvs F1mut-V10 was evaluated by three criteria: F1- and V-specific antibody titers, cytokine responses, and protection against Y. pestis CO92 challenge. Both the F1- and V-specific IgG antibodies  on day 88 post-first challenge, all the groups showed 100% protection except the T4-displayed F1mut-V10 group in which one mouse died (92% protection)  . All of Y. pestis, were vaccinated with alhydrogel adjuvanted F1mut-V, and F1mut-V10 as well as the T4 nanoparticle displayed F1mut-V and F1mut-V10 , given by the intranasal route, induced both TH1 and TH2 responses, providing better protection to mice against bubonic and pneumonic plague Y. pestis bacterium may require a balanced response that is generally not seen with the current F1-V vaccines Although humoral immune responses are critical for protection against plague, several studies have shown that cell-mediated immunity also plays important roles hallenge  and 11. Y. pestis infection in cynomolgus macaques as well as African Green monkeys, potentially leading to human clinical trials. Although the soluble F1mut-V vaccine adjuvanted with alum would be relatively easy to manufacture, the phage T4 nanoparticle-decorated F1mut-V vaccine offers certain advantages. First, the T4 formulation provided enhanced vaccine potency in small animal models. Second, the T4 vaccine would not require an extraneous adjuvant, and third, additional antigens from other biodefense pathogens, such as the protective antigen (PA) from Bacillus anthracis could be incorporated into the same formulation generating a dual vaccine against both inhalation anthrax and pneumonic plague. Our recent study demonstrated that the T4 displayed PA provided complete protection to Rhesus macaques against aerosol challenge with Ames spores of B. anthracisThere has been a considerable urgency to develop a recombinant plague vaccine, but several concerns precluded licensing of current formulations. Our studies have established that the F1mut-V recombinant vaccine is efficacious and easily manufacturable and should be seriously considered as a next generation plague vaccine. Future studies would include preclinical evaluation of protection against This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas Medical Branch, Galveston, TX,  and The Catholic University of America .E. coli expression vector pET28b  was used for recombinant plasmid construction. The template DNAs containing Y. pestis F1, V, or YscF were kindly provided by Dr. Richard Borschel from the Walter Reed Army Institute of Research . E. coli XL-10 Gold cells  were used for the initial transformation of clones. The plasmid DNAs were then re-transformed into E. coli BL21 (DE3) RIPL  for expression of recombinant proteins. The Hoc− Soc− phage T4 was propagated on E. coli P301 and purified by CsCl gradient centrifugation.The T7 promoter containing 2-terminus. The YscF mutant, YscF35/67, which contained point mutations at aa 35 (Asn to Ser) and 67 (Ile to Thr) was amplified by overlap PCR 2-terminal 14 aa residues were deleted. This PCR product was used as a template for the second round of PCR using a forward primer containing NheI restriction site and a reverse primer containing the NH2-terminal 14 aa residues and XhoI restriction site. The PCR fragment was then inserted into NheI and XhoI linearized pET28b vector.The DNA encoding F1, V, or YscF were amplified by PCR using primers containing appropriate restriction site(s) . The PCR products were purified, digested with appropriate restriction enzymes, and ligated with pET-28b vector DNA digested with the same restriction enzymes. The resulting plasmids had F1, V, or YscF coding sequences fused in-frame with the 23 aa vector sequence containing a hexa-histidine tag at the NH2-terminus of F1. The F1mut-V10 was amplified by overlap PCR using F1mut-V as the template and the mutated DNA was inserted into the NheI and HindIII linearized pET28b vector.To construct F1mut2 in which aa residues 15 to 21 were duplicated at the COOH-terminus, a reverse primer with a 5′-tag corresponding to the 15 to 21 aa sequence and XhoI restriction site was used for PCR amplification. The F1mut2 fragment was then inserted into NheI and XhoI linearized pET28b vector. To construct F1-V recombinants, V was first amplified and inserted into BamHI and HindIII linearized pET28b vector to generate the pET-V clone. F1 and F1mut2 were amplified with primers containing NheI and BamHI restriction sites, digested with NheI and BamHI, and ligated with the pET-V vector DNA digested with the same restriction enzymes. The resulting F1-V and F1mut-V plasmids contained F1 or F1mut in-frame fusion with V and a 23-aa vector sequence containing the hexa-histidine sequence at the NH2-terminus of RB69 Soc and also the flanking vector sequences containing two hexa-histidine tags at both NH2- and COOH-termini. The F1mut-V-Soc was then fused with YscF by overlap PCR with a two aa linker, GS, between Soc and YscF. All of the clones were sequenced  and only the clones containing 100% sequence accuracy were used for protein purification. The primer sequences used and clones generated in these studies will be available upon request.T4 Soc gene or RB69 Soc gene was fused with V, F1, or YscF with two aa (GS) linker by overlap PCR and the amplified DNA was inserted into the pET28b vector. The fused products V-T4 Soc, F1-T4 Soc, V-RB69Soc, and F1-RB69 Soc were further fused to YscF by overlap PCR to generate V-Soc (T4 or RB69)-YscF and F1-Soc (T4 or RB69)-YscF. Two aa residues, GS, were used as a linker between Soc and YscF. To construct F1-V-Soc clones, RB69 Soc gene was first amplified with end primers containing HindIII and XhoI restriction sites and inserted into the HindIII and XhoI linearized pET28b vector. This clone was then linearized by digestion with NheI and HindIII restriction enzymes. F1mut-V and F1mut-V10 DNAs were amplified by using the end primers containing NheI and HindIII restriction sites and inserted into the above plasmid. The resulting clones contained F1mut-V or F1mut-V10 fused in-frame to the NH+ T cell epitope prediction, F1 protein sequence was screened against 14 human MHC-II alleles. Peptides identified as positive ones by at least one predictor method were considered as potential CD4+ T cell epitopes. For the CD8+ T cell epitope prediction, F1 was screened against 57 human MHC-I alleles. Peptides identified as positive by at least one ensemble predictor approaches were considered to be potential CD8+ T cell epitopes. Default values were used for both the T cell epitope predictions.The structural models of F1, V, YscF, and T4 phage nanoparticle  were conE. coli BL21 (DE3) RIPL cells harboring various plague recombinant plasmids constructed as above were induced with 1 mM IPTG for 1 to 2 h at 30°C. The cells were harvested by centrifugation at 4,000 g for 15 min at 4°C and the pellets were resuspended in 50 mM Tris-HCl (pH 8.0). Solubility analysis was carried out using bacterial protein extraction reagent (B-PER) . The cells were lysed with B-PER and centrifuged at 12,000 g for 10 min. The soluble supernatant and insoluble pellet fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) as follows. The samples were boiled in a buffer containing SDS and β-mercaptoethanol, and were electrophoresed on a 12% or 15% (w/v) polyacrylamide gel. Since the protein aggregates will be dissociated into monomers under these conditions. The molecular weight differences observed in The g for 20 min. The supernatants were filtered through 0.22 µm filters  and loaded onto 1 ml HisTrap column  pre-equilibrated with 20 ml of binding buffer. After washing with the binding buffer containing 50 mM imidazole, the proteins were eluted with 20–500 mM linear imidazole gradient. The peak fractions containing the desired protein were concentrated by Amicon Ultra-4 centrifugal filtration . The proteins were further purified by gel filtration on Hi-load 16/60 Superdex 200 column  in a buffer containing 20 mM Tris-HCl, pH 8.0 and 100 mM NaCl. The peak fractions containing the purified proteins were concentrated and stored at −80°C. The native F1 recombinant proteins were purified from the pellet containing the insoluble inclusion bodies. The pellet was dissolved in the binding buffer containing 8 M urea and loaded onto 1 ml HisTrap column  pre-equilibrated with the same buffer. The proteins were renatured by washing the column with a decreasing urea gradient (8 to 0 M) in the binding buffer. The bound proteins were then eluted with 20–500 mM linear imidazole gradient. If necessary, the peak fractions from the HisTrap column were concentrated by Amicon Ultra-4 centrifugal filtration (10 kDa cut-off). The proteins were further purified by gel filtration on Hi-load 16/60 Superdex 200 column as described above.For protein purification, the cells were resuspended in binding buffer  containing proteinase inhibitor cocktail . The cells were lysed by French press  at 12,000 psi and the soluble fractions containing the His-tagged fusion proteins were isolated by centrifugation at 34,000 Y. pestis antigens from E. coli; F1, LcrV, YscF, and F1mut-V, were determined using Endosafe PTS system . This system consists of a handheld spectrophotometer and utilizes FDA approved disposable cartridges. At least three batches of each antigen were tested. The endotoxin levels ranged from 0.05 to 0.8 EU/ml, substantially lower than the maximum recommended in gene vectors and subunit vaccines, 10 and 20 EU/ml respectively, for preclinical research The levels of lipopolysaccharide (LPS) contamination in the purified recombinant In vitro binding of plague-Soc fusion protein on Hoc− Soc− T4 phage was carried out as previously described 10 phage particles were sedimented for 45 min at 34,000 g in LoBind Eppendorf tubes and resuspended in phosphate-buffered saline (PBS) buffer (pH 7.4). Various Soc fusion proteins were incubated with the resuspended Hoc− Soc− phage at 4°C for 45 min. The phage particles were sedimented at 34,000 g for 45 min and the supernatant containing the unbound protein was discarded. The phage pellet containing the bound plague antigen(s) was washed twice with excess buffer containing 20 mM Tris-HCl pH 8 and 100 mM NaCl. The final pellets were resuspended in PBS buffer (pH 7.4) and analyzed by SDS-PAGE. The gels were stained with Coomassie Blue R250  and the protein bands were quantified by laser densitometry . The density of Soc fusion protein, gp23*, and gp18  bands were determined for each lane separately and the copy number of bound plague antigen molecules per capsid was calculated using the known copy numbers of gp23* (930 molecules per capsid) or gp18 (138 molecules per capsid). A saturation binding curve relating the number of bound plague protein-Soc molecules per capsid (Y) and the concentration of unbound protein in the binding reaction (X) was generated by SigmaPlot software. The apparent Kd (association constant) and Bmax (maximum copies of Soc fusion protein bound per capsid) were determined using the equation Y = BmaxX/(Kd+X) as programmed in the SigmaPlot software.Y. pestis CO92 BEI strain 50. Animals were monitored and recorded twice daily for mortality or other symptoms for 48 to 88 days. The animals that survived were re-challenged intranasally at 48 or 88 days post-first challenge with the indicated LD50 and monitored twice daily for a further 48 days.Six to eight weeks female Balb/c mice (17–20 g) were purchased from Jackson Laboratories  and randomly grouped and acclimated for 7 days. Equivalent amounts of plague immunogen molecules, either soluble or phage-bound, were used for each immunization. For immunization of soluble antigens, the purified proteins (10 µg/mouse/immunization) were adsorbed on alhydrogel  containing 0.19 mg of aluminum per dose. For the T4 displayed antigens, the phage particles were directly used without any adjuvant (10 µg of plague antigen/mouse/immunization). On days 0, 21 and 42, mice were vaccinated via the intramuscular route. Alternate legs were used for each immunization. Blood was drawn from each animal on days 0 (pre-bleeds), 35 and 49 and the sera obtained were stored frozen at −70°C. On day 56, mice were intranasally challenged with 50 of Y. pestis CO92 BEI strain and were monitored twice daily for 30 days and clinical symptoms of disease and survival recorded.Female Brown Norway rats (50–75 g) were purchased from Charles River . Upon arrival, animals were weighed and randomized into the treatment groups and were acclimated for several days before manipulation. The plague immunogens were prepared as described above. On days 0, 21 and 42, rats were vaccinated via the intramuscular route with 15 µg antigen in 50 µl PBS buffer. Alternate legs were used for each immunization. On day 56, animals were intranasally challenged with 5,000 LD2SO4 and the absorbance was read at 450 nm using an ELISA reader. For IgG subtypes, horseradish peroxidase-conjugated goat anti-mouse IgG1 or IgG2a secondary antibodies were used.The IgG titers were determined by ELISA. Briefly, 96-well microtiter plates  were coated with 10 ng/well of purified F1, V, YscF, F1-V or F1mutV antigen at 4°C overnight. Following blocking and washing, sera from naïve and immunized mice were serially diluted and incubated with the affixed antigens for 1 h at room temperature. Following several washes, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody was added to the wells at a dilution of 1∶10,000. After incubation for 1 h at room temperature, the unbound antibody was removed and the wells were washed several times and the TMB  substrate was added. Following a 20 min incubation to develop the color, the reaction was quenched by the addition of 2 N H6 cells/ml and 1 ml of lymphocytes seeded into each well. Triplicate cultures from each group were stimulated with purified F1-V (10 µg/ml). Additional control stimulators included medium only and concanavalin A (5 µg/ml). After approximately 48 h incubation at 37°C in a humidified (5% CO2 in air) incubator, culture supernatants were collected. Cytokines were measured using a multiplex assay . The results were analyzed in Prism and the statistical significance was determined by one way ANOVA with Bonferroni correction.Seven days after the second boost (day 49), mice were sacrificed and spleens were harvested to prepare splenocytes using the lymphocyte separation medium. The isolated lymphocytes were adjusted to ∼5×10caf1) , lcrV , yscF , and soc (RB69Soc) .F1 capsule antigen (Text S1+ T cell epitopes. CD8+ T cell epitopes were predicted using MetaMHC (http://www.biokdd.fudan.edu.cn/Service/MetaMHC.html) with default values. Peptides identified as positive by at least one ensemble predictor approach were considered to be potential CD8+ T cell epitopes. Highlighted cells indicate high ranking scores that predict a potential CD8+ T cell epitope. Table S2. The predicted CD4+ T cell epitopes. CD4+ T cell epitopes were predicted using MetaMHC (http://www.biokdd.fudan.edu.cn/Service/MetaMHC.html) with default values. Peptides identified as positive by at least one ensemble predictor approach were considered to be potential CD4+ T cell epitopes.Table S1. The predicted CD8(DOC)Click here for additional data file.


Candida albicans is an opportunistic pathogen that causes acute disseminated infections in immunocompromised hosts, representing an important cause of morbidity and mortality in these patients. To study the genetic control of susceptibility to disseminated C. albicans in mice, we phenotyped a group of 23 phylogenetically distant inbred strains for susceptibility to infection as measured by extent of fungal replication in the kidney 48 hours following infection. Susceptibility was strongly associated with the loss-of-function mutant complement component 5 (C5/Hc) allele, which is known to be inherited by approximately 40% of inbred strains. Our survey identified 2 discordant strains, AKR/J  and SM/J , suggesting that additional genetic effects may control response to systemic candidiasis in these strains. Haplotype association mapping in the 23 strains using high density SNP maps revealed several putative loci regulating the extent of C. albicans replication, amongst which the most significant were C5  and a novel effect on distal chromosome 11 . Compared to other C5-deficient strains, infected AKR/J strain displays a reduced fungal burden in the brain, heart and kidney, and increased survival, concomitant with uniquely high levels of serum IFNγ. C5-independent genetic effects were further investigated by linkage analysis in an [A/JxAKR/J]F2 cross (n = 158) where the mutant Hc allele is fixed. These studies identified a chromosome 11 locus , and a chromosome 8 locus , both initially detected by haplotype association mapping. Alleles at both loci were inherited in a co-dominant manner. Our results verify the important effect of C5-deficiency in inbred mouse strains, and further identify two novel loci, Carg3 and Carg4, which regulate resistance to C. albicans infection in a C5-independent manner. Candida albicans is an opportunistic fungal pathogen that exists commensally in the gastrointestinal and genitourinary tracts of healthy individualsCandida species constitute the fourth most common causative agent of nosocomial bloodstream infections, and are associated with significant attributable mortality in both adults and children (47% vs. 29%)albicans Candida species, Candida albicans remains the most common isolate recovered from bloodstream infections worldwide, with the frequency of occurrence ranging from 37% to 70%C. albicans infection in humansCandida infectionsC. albicans is under a complex genetic control that affects onset of infection, type and severity of disease developed and associated pathologies , and extent of immune response elicitedC5 allele, which causes susceptibility to infection with C. albicans, as well as Cryptococcus neoformans−/− animals which show impaired fungal clearance and higher mortality than wild type controls when infected with C. albicans or C. glabrataIn mouse models of infection, response to C5 allele on fungal load and survival time of C. albicans infected mice may be further modulated by genetic background effectsC. albicans infection has pointed to C5-unrelated genetic loci, temporarily given the appellation Carg1 and Carg2C. albicans infection. With the aim of identifying such additional gene effects, we have herein phenotyped a total of 23 phylogenetically distant inbred strains of mice for susceptibility to C. albicans. These studies have identified two strains (AKR/J and SM/J) that show discordant genotype/phenotype relationships with respect to C5 status and susceptibility to infection. Haplotype association mapping in the 23 inbred strains using high density SNP maps, together with genetic linkage analyses in informative crosses derived from discordant strains, have uncovered two C5-independent genetic effects and loci controlling fungal replication in the host, and mapping to chromosomes 8 (Carg3), and 11 (Carg4).Studies in inbred strains and in AcB/BcA recombinant congenic strains have suggested that the deleterious effect of the C. albicans organisms in target organs during disseminated infection, we surveyed 23 strains from the panel of 36 commonly used inbred mouse strains represented in the Jackson Mouse Phenome Database. These strains have been selected to be genetically diverse, on the basis of their phylogenetically distinct breeding background, and thus likely to be representative of the natural allelic pool. The mice were challenged intravenously with a low dose of C. albicans SC5314 and the fungal replication was assessed in the kidney 48 h following infection. We observed wide differences in the extent of C. albicans colonization and replication in kidneys of these inbred strains (10CFU = 2.3±0.5) such as in the BPL/1J strain, to approximately 10 000-fold greater number of fungi in the highly susceptible A/J (log10CFU = 6.0±0.1), along with a number of strains showing a wide range of intermediate phenotypes. To determine the impact of the C5 locus and its mutant Hc allele on the response to C. albicans infection in these strains, we established the C5 genotype of the 23 strains (10CFU>5.1) consisting of 7 strains and a resistant group (log10CFU<4.2) consisting of 16 strains. Using this arbitrary segregation, we noted a very strong effect and near perfect correlation of C5 allelic status and extent of replication of C. albicans in kidney with two notable exceptions (10CFU = 5.2±0.4) when compared to the reference B6 strain (log10CFU = 4.1±0.5) or other C5-sufficient strains. AKR/J mice are C5-deficient 10CFU counts at 3.9±0.4 similar to that seen in the resistant B6 strain, and clearly distinct from that seen in the C5-deficient strains group (log10CFU>5.1).To identify novel C5-independent genetic effects regulating the proliferation of  strains , ranging strains . We alsoceptions . DespiteC5 mutant allele on the response of inbred strains to disseminated C. albicans infection, the wide variation of CFU counts observed within mouse strains bearing wild type C5 alleles and the presence of two clearly discordant strains , together strongly suggested the presence of C5-independent genetic effects in the cohort of 23 strains tested.Although these results confirm the critical impact of the C. albicans, we used the strain distribution pattern (kidney CFU counts), and high density SNP datasets available for the 23 strains (Mouse HapMap SNP data) to perform whole genome association mapping. We utilized a statistical method EMMA −5) on chromosomes 1, 2, 4, 6, 7, 8, 11, 12, and 15 (−8) which identified SNPs on chromosomes 2 and 11 as the most highly associated with CFU counts. As expected, the top 3 significant SNPs  are situated between 33.74 and 33.96 Mb on chromosome 2, and correspond to the C5 gene located less than 1 Mb away. Repeating the EMMA analysis after removing phenotypically discordant AKR/J and SM/J strains, increases the significance of the Chr. 2 (C5) locus dramatically , PWD/PhJ (M. m. musculus), and the CAST/EiJ (Mus castaneus). The next most significant association  was detected on the distal portion of chromosome 11 (98.87 Mb). Haplotype analysis of the corresponding Chr. 11 shows a haplotype block less conserved in inbred strains than the Chr. 2 haplotype, including multiple possible recombination events within the haplotype map . We have previously established that C5-deficiency is associated in A/J mice with a striking cardiac phenotype, prior to and during C. albicans infection C. albicans replication may be linked to such a heart-specific effect of C5-deficiency present in AKR/J. Together, these results strongly suggest that AKR/J mice are able to contain C. albicans replication in all organs, despite C5-deficiency.The differential susceptibility of A/J and AKR/J mouse strains to C. albicans,  and monitored in these animals: a) organ CFUs, b) kidney function as measured by BUN (blood urea nitrogen), and c) survival  as done previously and monitored survival daily. All A/J mice succumbed after 24 hours (highly susceptible), whereas the median survival time (MST) for AKR/J mice was 3 days , while MST for resistant B6 mice was 4 days.We further challenged AKR/J and A/J mice with several infectious doses of survival . With reC. albicans infection C. albicans infection using the BD™ CBA Flex system  at 24 and 48 h following x system . As expeC. albicans resistance of AKR/J mice was investigated by phenotyping mice from an informative F2 cross generated between resistant AKR/J and susceptible A/J progenitors, and in which all mice are fixed for C5 deficiency. [A/JxAKR/J]F1 hybrids showed an intermediate level of susceptibility with respect to kidney fungal load  for chromosome 8 QTL and Carg4, for chromosome 11 QTL. The Bayesian 95% credible intervals were determined to be 53.6–96.9 Mb for Carg3 and 70.7–103.8 Mb for Carg4, whereas the peak LOD scores were identified at 75 Mb (peak SNP: rs3722665) and 98.8 Mb (peak SNP: rs3661058), respectively. Both Carg3 and Carg4 loci were present amongst the initial SNP associations uncovered by EMMA analysis (−5), however, only Carg4 passed the stringent Bonferroni correction. These results confirm results of the haplotype mapping studied in the 23 inbred strains, with respect to the effect of the Carg4 locus on response to disseminated C. albicans infection. They also suggest that the Carg4 locus strongly contributes to the noted resistance of AKR/J mice, acting independently of the C5 locus, for which susceptibility alleles are fixed in the analyzed AKR/JxA/J cross.The inheritance and complexity of the C5-independent genetic control to gal load  comparedfections . The kidfections  and the ]F2 mice  show a n]F2 mice . This an]F2 mice . These lanalysis  and wereCarg3 and Carg4 increased the LOD score to 11.5, suggesting an additive effect which explains 28% of phenotypic variance observed. This additive effect is clearly illustrated when the extremities of the F2 distribution are segregated according to the more significant Carg4 locus . Although a few select resistant F2 animals carry the A/J allele at one locus, they consistently bear a non-A/J allele (AKR/J or heterozygous) at the other locus. The opposite is also true for susceptible F2 mice which inherited the AKR/J allele at either Carg3 or Carg4. Finally, to illustrate the overlap achieved by EMMA and linkage analysis, association scores for chromosome 11 and 8 are depicted along with respective LOD score traces  controlling fungal replication in the host. Subsequent genome scan in informative F2 mice generated between susceptible A/J and resistant AKR/J  progenitors identified two highly significant linkages on chromosomes 8  and 11 , as regulating permissiveness to C. albicans in these mice. Carg3 and Carg4 are novel loci that regulate susceptibility to C. albicans in a C5-independent fashion.We have used a representative panel of 23 phylogenetically distant inbred mouse strains to study the genetic control of susceptibility to acute and disseminated C. albicans in inbred strains. A noteworthy advantage of the EMMA algorithm is the avoidance of inflation of false positives due to a kinship matrix that corrects for genetic relatedness amongst inbred strainset al.Carg3 (LOD = 3.95) and confirmed the chromosome 11 locus   . In factectively  stronglyC. albicans infected mice correlate with kidney damage C. albicans. The phenotypic response to infection in C5-deficient AKR/J mice with respect to serum cytokine profiles is clearly distinct from that of C5-deficient A/J mice, but rather resembles that of resistant and C5-sufficient C57BL/6J strain, with one notable exception. Indeed, infected AKR/J mice show strikingly elevated levels of serum IFNγ compared to either resistant C57BL/6J or susceptible A/J. Although we cannot yet establish that elevated serum IFNγ seen in AKR/J are solely or partly responsible for resistance in this strain, a protective role for this cytokine in C. albicans infection has been established. Indeed, mice deficient for IFNγC. albicans, and passive administration of IFNγin vitro studies have also shown that IFNγ potentiates phagocytosis and killing of C. albicans by neutrophilsCarg3 and Carg4 loci.Recently, it was demonstrated that elevated levels of IL-6, MIP-1β, and mostly KC in kidney extracts of et al. recently used chromatin immunoprecipitation and DNA sequencing to map all functional STAT1 binding sites (and associated genes) which are induced by IFNγ treatment of human HeLa S3 cells Carg3 and Carg4 for the presence of positional candidates that may be bound by STAT1 in response to IFNγ. The minimal genetic interval inferred by linkage analysis at Carg3 extends over 40 Mb, while that at Carg4 is 30 Mb. As both regions are extremely gene rich, we restricted the size of the genomic candidate regions by combining linkage analysis and the haplotype association mapping results. Therefore, we prioritized for analysis blocks of 10Mb centered around the SNPs showing the highest scores in the haplotype association studies for both loci (Carg3) and 147 such genes (Carg4)  elements and also interferon-stimulated response elements (ISREs)oth loci . This an (Carg4)  and S3. Carg3 interval fulfill both criteria: Adcy7, Dnaja2, Gab1 and Inpp4b. Adcy7 is of particular interest for the following reasons. Adcy7 codes for an isoform of adenylate cyclase that is expressed at high levels and regulates the intracellular levels of cyclic adenosine monophosphate (cAMP) in macrophages, T cells and B cells Adcy7 expression is abundant in macrophages, stimulated by IFNγ, and associated with STAT-1 binding to the Adcy7 gene promoter  and made publically available. [A/JxAKR/J] F2 progeny were bred by systematic brother-sister mating of [A/JxAKR/J] F1 mice. All housing and experimental procedures were conducted under the guidelines of the Canadian Council of Animal Care and were approved by the Biotechnology Research Institute (BRI) Animal Care Committee (Protocol number: 08-SEP-I-017) and the Animal Care Committee of McGill University (Protocol number: 5618).Inbred strains 129S1/SvlmJ, A/J, AKR/J, BALB/cByJ, BPL/1J, BTBRT+tf/J, BUB/BnJ, C3H/HeJ, C57BL/6J, C57BLKS/J, CAST/EiJ, CBA/J, DBA/2J, DDY/JclSidSeyFrkJ, FVB/J, KK/HlJ, MRL/MpJ, MSM/Ms, NOD/ShiLtJ, NZW/LacJ, PWD/PhJ, SM/J, and SJL/J were obtained as pathogen-free mice at 8–12 weeks of age from the Jackson Laboratory  as part of a collaboration with the Mouse Phenome Database project. Survey data will be deposited in MPD  at 30°C. Blastospores were harvested by centrifugation, washed twice in phosphate-buffered saline (PBS), counted using a hemacytometer and resuspended in PBS at the required density. For experimental infections, mice were inoculated via the tail vain with 200 µl of a suspension containing 5×104C. albicans blastospores in PBS. In dose-response experiments, additional doses were tested: 5×104, 1×105, 3×105, and 6×105C. albicans. Forty-eight hours following infection (unless otherwise indicated), target organs were removed aseptically and homogenized in 5 ml of PBS. The homogenate was then serially diluted and plated on YPD-agar plates containing 34 µg/ml of chloramphenicol. The plates were incubated at 30°C for 24–48 hours and the colony-forming units (CFU) counted and expressed as log10CFU per organ. The maximum sensitivity for this assay was 100 CFU, and the animals displaying titers below the detection limit were assigned an arbitrary value of 100 CFU.5C. albicans blastospores in PBS. Mice were closely monitored for clinical signs such as lethargy, hunched back, and ruffled fur. Mice exhibiting extreme lethargy were deemed moribund and were euthanized.For the survival study, mice were injected intravenously with 200 µl of a suspension containing 3×10Mice were anesthetized and exsanguinated by cardiac puncture. Serum was isolated by collection of blood, followed by centrifugation and storage at -80°C until used to measure cytokine levels. To determine the levels of cytokines in the circulation, 12.5 µl of serum was analyzed using the BD™ CBA Flex sets according to the manufacturer’s instructions. Fluorescence levels were recorded using the BD™ LSRII flow cytometry system  using BD FACSDiva acquisition software and the data analysis was carried out using the FCAP Array software.At 24 h or 48 h, mice were exsanguinated by cardiac puncture under anesthesia, and blood was collected in microtubes with separation gel . Serum was isolated by centrifugation and stored at −20°C until used for blood urea nitrogen (BUN) determination. A commercially available kit that allows quantitative urease/Berthelot determination was used to measure BUN levels (Sigma).C. albicans elements and counterstained with hematoxylin to visualize immune cells infiltration. Stained sections were examined under a light microscope at 100X and 400X magnification and photographed.Whole kidneys were fixed in 10% neutral buffered formalin, dehydrated in ethanol/xylene and embedded in paraffin, as described previously www.informatics.jax.org) and used for gap filling and fine mapping by a standard PCR-based method employing (α-32P) dATP labeling and separation on denaturing 6% polyacrylamide gels.As described previouslyQTL mapping was performed using Haley-Knott multiple regression analysisThe detailed algorithm underlying the efficient mixed-model for association mapping have been previously published2 statistic. These data were analyzed using GraphPad Prism 4.0 statistical software. P-values<0.05 were considered significant.An unpaired, two-tailed Student’s t-test was used to establish significance of differences in mean CFU per organ, BUN levels, and cytokine concentrations between different mouse strains. Survival of AKR/J mice was analyzed by a Log-rank test and survival fractions were compared using the χFigure S1Genome-wide association mapping without discordant AKR/J and SM/J strains. EMMA analysis was conducted as described earlier  and the Bonferroni multiple testing correction threshold was calculated to be 7.87×10−8 (dashed line).lier see  while om(PDF)Click here for additional data file.Table S1In silico identified loci controlling response to C. albicans infection in inbred mouse strains. SNPs that have passed the Bonferroni cutoff  are shaded in gray.(PDF)Click here for additional data file.Table S2Candidate genes in the Carg3 region. A total of 59 genes containing IFNγ-inducible STAT1 binding sites and their overall mRNA expression (>2X) upon IFNγ stimulation in Hela cells are represented. Genes considered for further prioritization had a high (>20) ChIP-Seq peak height and a significant (>2X) gene expression. N/A designation in the gene expression column was given for genes that were not represented on the microarray.(PDF)Click here for additional data file.Table S3Candidate genes in the Carg4 region. A total of 147 genes containing IFNγ-inducible STAT1 binding sites and their overall mRNA expression (>2X) upon IFNγ stimulation in Hela cells are represented. Genes considered for further prioritization had both high (>20) ChIP-Seq peak height and a significant (>2X) gene expression. N/A designation in the gene expression column was given for genes that were not represented on the microarray.(PDF)Click here for additional data file.


Pichiapastoris is one of the most widely used expression systems for the production of recombinant secretory proteins. Its universal application is, however, somewhat hampered by its unpredictable yields for different heterologous proteins, which is now believed to be caused in part by their varied efficiencies to traffic through the host secretion machinery. The yeast endoprotease Kex2 removes the signal peptides from pre-proteins and releases the mature form of secreted proteins, thus, plays a pivotal role in the yeast secretory pathways. In this study, we found that the yields of many recombinant proteins were greatly influenced by Kex2 P1' site residues and the optimized P1’s amino acid residue could largely determine the final amount of secretory proteins synthesized and secreted. A further improvement of secretory yield was achieved by genomic integration of additional Kex2 copies, which again highlighted the importance of Kex2 cleavage to the production of recombinant secretory proteins in Pichia yeast. Pichiapastoris is one of the most commonly used expression hosts for production of heterologous secretory proteins [AOX1), high levels of protein products being secreted into almost protein-free media as well as its capacity of carrying out correct folding and post-translational modification for mammalian proteins [Pichiapastoris genome [Protein based biopharmaceuticals make up the largest and fastest growing part of global top selling drugs ,2. Picproteins , thanks proteins -8. Genet genome -11, tran genome -15, and  genome , as well genome -19, prom genome -24, has  genome . Althoug genome -28, for  genome . HoweverKEX2 gene encodes a Ca2+ dependent serine endoprotease [in vivo positive correlation between Kex2 cleavage and yeast secretory rate has been reported [in vitro enzymatic characterizations with short synthetic peptide as substrates [in vivo.The yeast protease ,30 whichprotease ,32  and several mammalian proteins were tested in this library system , D-sorbitol, D-biotin and BCA protein concentration assay kit were purchased from Invitrogen . Tryptone and yeast extract were purchased from Oxoid . Polyethylene glycol (PEG) 3350 and lithium chloride (LiCl) were purchased from Sigma-Aldrich . Sonicated single stranded salmon sperm DNA was purchased from Genmed . Zeocin was purchased from Invivogen . Steady-Glo® Luciferase Assay System was purchased from Promega . Plasmid miniprep kit, DNA recovery/purification kit, pMD20-T cloning vectors, restriction endonucleases, DNA polymerases for PCR reaction and T4 DNA ligase were purchased from Takara . Primers synthesis and DNA sequencing service were provided by Invitrogen . Escherichia coli TOP10 single colonies were selected on normal (for pMD20-T cloning) or low salt (for pPICZαA cloning) LB agar plates  or 0.5%  NaCl and 1.5% agar) with corresponding antibiotics. Newly generated Pichiapastoris transformants were initially selected on YPD plates  with 100 µg/ml zeocin, then on YPD plates with increasing doses of zeocin  to determine the copy number of integrants. For methanol induced expression, the Ppastoris.  recombinants were first grown in BMGY , 1.34% YNB, 4×10-5% biotin, 1% glycerol) medium to reach higher biomass and then induced in BMMY medium which contains the same ingredients as BMGY except the replacement of glycerol with methanol, as detailed in EasySelectTMPichia expression kit user manual.Venus coding DNA sequence (CDS), followed by kanamycin resistance gene, was amplified with primers (forward primers which covered the P1’ site were listed on 5’-GGCTAGCGGCCGCAGACATGATAAGATACATTGATGAG-3’ which complements the 3’ terminus of kanamycin resistance gene) to place all 20 amino acids (codons were chosen according to the yeast preference [eference ) at P1’ 5’-GGAATTCCTCGAGATGGAAGACGCCAAAAACATAA-3’ as the forward primer and 5’-GGCGGCCGCTAGCACGGCGATCTTTCCGCCCTTC-3’ as the reverse primer and sub-cloned into pMD20-T plasmid and sequenced. Then the inserted plasmid was digested with EcoRI and NotI and the inserted fragments were recovered and ligated with vector backbone fragments originated from the Venus vector library.For construction of luciferase version of the library , the couFor construction of other recombinant mammalian proteins version of the libraries , the cou5’-GGCCATTACGGCCAAGCTTGGCCAGGGCGGCCCACGTCCGACGGCGGCCCACGG-3’ as the forward primer and 5’-GGCCGCCCTGGCCAAGCTTGGCCGTAATGGCCGGTTTAGTTCCTCACCTTGTCG-3’ as the reverse primer. Second, the Ppastoris.  Kex2 CDS was cloned from yeast genome with 5’-CGGATCCACCATGTATTTGCCAGCACTTCGCTTAGC-3’ as the forward primer and 5’-GCTCGAGCAATGCCGCACGTTTGGGATGTTCATTAG-3’ as the reverse primer, sequenced and inserted into a plasmid to fuse the flag tag to C-terminus of the CDS and SfiI sites at both ends of this cassette. After elimination the endogenous SacI site with synonymous point mutation, Kex2 CDS was then subcloned into the Zeo(R) deleted S-Venus and G-Venus between SfiI sites. Finally, Zeo(R) was reintroduced into these plasmids at BamHI site to the 3’ end of AOX1 transcription terminator.For plasmids to introduce additional Kex2 copies into yeast genome , we tookPpastoris.  X-33 with the lithium chloride transformation method described in EasySelectTMPichia expression kit user manual (Invitrogen). Transformants were initially grown on YPD plates supplemented with 100 µg/ml zeocin. After integration of the plasmid into the yeast genome was confirmed by colony PCR, resulting colonies were transferred to YPD plates with 200, 500 and 1000 µg/ml zeocin for determination of the copy number of integrants. The subsequent comparisons of secreted proteins were only made between transformants with approximately the same copy numbers as determined by the same concentration range of drug resistance against zeocin.The yeast expression library vectors were linearized by SacI digestion and transformed into TMPichia expression kit user manual to express recombinant Pichia pastorish has been followed. Briefly, the selected colonies were initially cultivated (shaking vigorously at 250 rpm) in BMGY medium at 28-30°C until the value of OD600 reached 2 approximately. After centrifugation and removal of BMGY, cell pellets were re-suspended in BMMY to an OD600 of 1 to induce expression. The volume of the culture should be no more than 10-30% of the total well/tube/flask volume to ensure sufficient aeration. Methanol was added to a final concentration of 1% every 24 hours to maintain induction. Yeast culture media were sampled and assayed every 24 hours. Small-scale cultivation and expression using 96-deep-well plates  were carried out as previously described [The experimental protocol from EasySelectescribed ,43 whene600 and the values were used to normalize fluorescence or luminescence data. For Venus fluorescence assay, the cells were sprung down and 80 µl supernatant was added to each well of 96-well solid bottom black plates  and the signal measured under an excitation spectrum of 515 nm and an emission spectrum of 528 nm. For luminescence assay, 50 µl supernatant was added with equal volume of Steady-Glo® reagent in each well of 96-well solid bottom white plates  and the signal measured on a Veritas TMmicroplateluminometer from Turner Biosystems .Before these assays, 100 µl yeast culture medium from each sample was measured for ODThe yeast cell lysis and cellular total protein extraction for SDS-PAGE and western blotting were carried out as described previously . The yeaPpastoris.  strain harboring the most productive P1’-stem cell factor  at 4°C overnight, filtrated with 0.22 µm filter, loaded onto a 5 ml HisTrapTM HP column  and washed with extract buffer. The protein was eluted with a gradient of 20-500 mM imidazole. The purified protein was sent for MS analysis for identification.Scale-up expression was carried out in a 2 L baffled flask. r V-SCF,  was cultP values were calculated with t-test (Student’s t-test); the actual P values of all statistical significances were given on the corresponding figure legends.Each fluorescent/luminescent value with an error bar was presented as mean±SEM representing the average of 5-6 individual colonies for the same vector with the same antibiotic resistance range for zeocin. Pichiapastoris strain X-33. The resultant yeast colonies grown on YPD plates containing antibiotic zeocin (100 µg/ml) were subjected to colony PCR to confirm transformation (data not shown), and transferred to YPD plates containing 200, 500 and 1,000 µg/ml zeocin to empirically estimate the copy number of the integrants. On average, 20-30 colonies derived from each vector were tested for their integrated copy number, from which 5-6 colonies for the given vector, with zeocin resistance ranging 200-500 and 500-1,000 µg/ml, respectively, were selected for subsequent analysis of their levels of secretion.The 20 yeast recombinant Venus/luciferase expression vectors were generated by PCR amplification and TA-cloning of Venus-Kana(R) fragments with all 20 AAs at P1’ site into the original yeast secretory expression vector pPICZαA . Both thCultivations were sampled every 24 hours till 120 hours post methanol induction. The supernatants of the yeast cultures were assayed for fluorescence (Venus) or luminescence (luciferase). As shown in To achieve the objective of increasing the yields of mammalian proteins with medical relevance, several such proteins were expressed using this methodology. The results showed that the yields of all the recombinant proteins tested could be enhanced by P1’ site replacement . The mosPpastoris.  hosts could further increase the secretory productivity. To this end, the Venus expression constructs with/without additional Kex2 copies were chosen, cultivated, sampled and expression levels determined. Western blotting analysis showed that introduction of additional Kex2 copies greatly increased the Kex2 expression in the yeast host cells (Kex2 copies in that G-Venus-kex2 nearly doubling the productivity (Ppastoris.  by optimizing P1’ site and increased Kex2 copies.Finally, we investigated whether additional copies of Kex2 in st cells . The fluuctivity . Taken tPichiapastoris as an eukaryotic protein expression system, the problem of inconsistent secretory productivity among different recombinant proteins, i.e. some proteins could reach extremely high yields [Ppastoris.  secretory productivity by optimizing the yeast convertase Kex2 cleavage.During decades of using h yields -28 whileh yields , based oin vitro enzymatic data [in vivo experiments might reflect the physiological situation of the Ppastoris.  host cells since influences of different P1’ residue on the secretory yields were variable. The most significant result of our study clearly demonstrated that the patterns and the extents of these variations depended on different proteins (Our study was carried out with the commonly used yeast secretory expression vector, i.e., pPICZαA. Through generating a library of vectors , yeast ttic data ,54, our proteins , Table 2Kex2 copies into the Pichia yeast genome has also been proved to significantly improve the secretory yields of recombinant secretory proteins regardless of the P1’ residues in our study (Ppastoris.  secretory productivity of recombinant proteins by making optimal use of Kex2 activity, which was accomplished by optimization of Kex2 P1’ site residue and/or introduction of additional Kex2 copies into yeast genome.Other than optimization of the P1’ residues, integration of additional constitutively expressing ur study , which d


MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects.The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy.In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype.While carrying a rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.These data suggest that subtle differences in receptor signaling conferred by rare  Obesity is a worldwide epidemic that contributes to comorbidities such as diabetes and heart disease Regulation of feeding and satiety, essential for maintaining healthy weight, occurs in the hypothalamus. In the fed state, insulin and leptin stimulate neurons expressing proopiomelanocortin (POMC) to release α-melanocyte stimulating hormone (α-MSH) and β-MSH MC4RMC4R, occurring in both lean and obese people at equal frequencies, have also been described MC4R variant I251L leads to better weight-loss and weight maintenance following RYGB I251L allele also resolved their T2D more quickly than patients with two copies of the MC4R reference allele MC4R variants have also been reported, primarily identified in cohorts of obese individuals et. al. grouped MC4R mutations into five classes: Class I are null mutations; Class II are mutations that cause localization defects; Class III are mutations that cause binding defects; Class IV are mutations that cause cAMP signaling defects; and Class V variants have no known defects MC4R variants have unknown defects, the analysis of each mutation to determine the in vitro signaling defects and their possible correlation with obesity phenotypes is necessary.The development of obesity, as well as the degree of weight-loss following RYGB surgery can be greatly impacted by genetic variants MC4R variants identified in a cohort of 1433 obese patients who underwent RYGB surgery. Of the fifteen variants, two (V103I and I251L) are common alleles occurring at equal frequencies in both lean and obese populations MC4R variants are rare; twelve were previously reported and one, G34A, is novel. Three of the rare variants were associated with poor weight-loss post-RYGB surgery , one exhibited reduced cell surface expression (R305Q) and two had reduced cAMP signaling (I137T and C326R). Despite the apparent differences in biochemical characteristics, no correlation was found between in vitro signaling and pre- or post-surgery metabolic phenotypes. Together these data point to the complex involvement of MC4R in obesity, metabolism and weight-loss.We report fifteen MC4R was determined for each sample and matched to clinical data obtained in a de-identified manner through a data broker. A cohort of 1433 patients ) who underwent primary Roux-en Y gastric bypass (RYGB) surgery has been previously described IR) was calculated as HOMAIR = (fasting plasma insulin×fasting plasma glucose)/22.5. DNA extraction from blood samples and MC4R gene sequencing was performed as described MC4R reference allele who are non-diabetic, T2D or T2D taking insulin were calculated by plotting the mean body mass index (BMI) and fitted with Hill plot curve as described 2) (±1), age- (±5 years), gender-, T2D status-, and whether they were taking insulin. Carrier and non-carrier patients' BMIs are plotted during the period 12 months prior to- and up to 84- months after RYGB. Three patients  could not be matched within these parameters, so the BMI range was extended to ±2–3 and/or the age range was extended to ±8 years.All procedures and patient information were collected under a protocol approved by the Geisinger IRB. All subjects gave written informed consent for this project. Genomic DNA was isolated from blood collected from patients. The sequence for Individual mutations were made with the Quickchange site-directed mutagenesis kit  in a 3x HA tagged MC4R in pcDNA3. The 3x HAMC4R was subcloned into pEF6V5:eGFP-CAAX-2A-mCherry (Addgene plasmid 26901) to create the HAMC4R2aGFP construct. A bungarotoxin binding site (MWRYYESSLEPYPD) 2. For transient transfections, cells were transfected with plasmids described above by Fugene  and used two days post-transfection.HEK293 cells  were cultured in MEM with 10% FBS at 37°C and 5% CO2, 2 MgCl2, 22 NaHCO3, 1.1 NaH2PO4, 2 glucose, pH 7.4, and labeled with 10 µg/mL Bungarotoxin (BTX) conjugated to Texas Red for 10–15 min at 4°C and rinsed again in Low K solution. Live cells were imaged on an inverted Olympus Spinning Disc confocal microscope with IPLab  image acquisition software and processed with ImageJ.Bungarotoxin Binding Site (BBS) tagged MC4R constructs (BBS-MC4R) were transfected into HEK 293 cells on poly-L-lysine coated glass bottom Fluorodishes . Cells were rinsed twice with Imaging Low K containing (in mmol/L): 25 HEPES, 114 NaCl, 2.2 KCl, 2 CaClApproximately 10,000 HA tagged construct transfected cells were added to wells of a poly-L-lysine coated 96 well plate. The following day, cells were washed with PBS and fixed with either methanol  or 4% paraformaldehyde (for surface expression). Cells were then blocked with 1% milk and incubated in peroxidase conjugated anti-HA antibody. The plate was washed with TBS-T three times and then incubated with 100 µL 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate  for 30 minutes. 100 µL of 1 mol/L sulfuric acid was added to each well to stop the reaction. Absorbance was then read at 450 nm on a Spectramax 250 plate reader. The absorbance from untransfected cells was first subtracted and then the cell surface labeled signal was plotted as a percentage of total signal  and then normalized to wild-type HA-MC4R expression for that experiment. Significant differences from wild-type were determined using one way ANOVA with Dunnet's post-hoc.HEK293 cells were transiently transfected with HAMC4R2aGFP or mutant HAMC4R2aGFP, where HA-MC4R and GFP are separated by the 18 amino acid 2a peptide sequence from the foot and mouth disease virus Cells were transfected with BBS-MC4R constructs and Exchange Protein directly Activated by cAMP (EPAC2)-camps sensor MC4R variants. Two of these variants, V103I and I251L, are common and occur in >1% of both lean and obese populations V103I and I251L carriers in the RYGB cohort and 451 lean subjects were similar S4F (11C>T), G34A (101G>C), H76R (227A>G), V95I (283G>A), T112M (335C>T), I137T (410T>C), F202L (606C>A), L207V (619C>G), L250Q (749T>A), V253I (757G>A), S295P (883T>C), R305Q (914G>A), C326R (976T>C) are considered rare, and occurred in just eighteen patients. Twelve of these variants have been reported previously S4F, H76R, V95I, I137T, L250Q, and C326RT112M, F202L, L207V, V253I, S295P and R305QG34A variant has not been previously reported. Notably, no rare variants in MC4R were found in our lean cohort.We sequenced the MC4R gene of 1433 patients who underwent primary RYGB. A total of eighty patients had fifteen distinct MC4R variant of each patient and their corresponding body mass index (BMI), T2D status pre- and post- surgery, hemoglobin A1c (HbA1c) , HOMAIR (homeostatic model assessment for insulin resistance) values, and pre-surgery blood pressure are detailed in MC4R reference allele MC4R variants lost weight following RYGB . To assess whether rare alleles of MC4R are associated with weight-loss outcomes following RYGB, we matched each patient carrying a rare MC4R variant with patients with the reference sequence for gender, age, starting BMI, and T2D status   were indistinguishable from matched controls , where excess body weight is defined as weight above BMI of 25. Using either one of these criteria, patients carrying V95I, I137T and L250Q did not achieve the same weight loss as others. However, the overall odds of achieving the same RYGB results, %BMI loss or %EBWL as defined above, were not different for patients carrying rare variants or non-carriers.The ing RYGB . Patientinsulin)  and 2. Tcontrols . Similarcontrols . Insuffipatients . Interesin vitro. For each of the fifteen MC4R variants in the RYGB cohort, we analyzed the cell surface localization by ELISA and imaging of cell surface labeled receptors. Approximately 40% of total MC4R was located at cell surface and all mutants showed similar percent cell surface expression. Only the R305Q mutation displayed a small but significant decrease in cell surface expression compared to the wild-type receptor  50 for cAMP production  or signaling (C326R) had no significant phenotypic consequences after RYGB. We conclude that carrying a rare variant of MC4R, while associated with obesity, does not affect weight-loss after gastric bypass surgery. This is consistent with reports of weight-loss in rodents after RYGB where only complete loss of MC4R function had a significant effect on weight-loss We and others have reported that, in general, carrying an These findings compel us to propose a dual role of MC4R in obesity and clinical outcomes after gastric bypass:MC4R variants may not play a significant role in weight-loss following RYGB, similar to the effect seen in heterozygous Mc4r mice Mc4r null mice fail to lose weight after RYGB I251L allele, which in vitro has increased basal activity Mc4r knock-out mice normalized weight-loss and insulin resistance in mice after gastric bypass surgery Post-RYGB weight-loss provides a short-term measurement in a more controlled setting, due to the physical influence of the procedure and the clinical follow-up conducted post-surgery. Our data show that modest deleterious effects of MC4R variants and obesity. Our data corroborate these findings since we only found rare variant carriers in the obese population Mc4r mice develop obesity on high fat diet or even regular chow; however, on reduced fat diet these mice do not gain significant weight compared to their wild-type littermates MC4R rare variant carriers can result in significant weight-loss, but an increased effort is needed to stay on a weight-loss regimen MC4R allele lost 6.6±0.9% of their initial weight, while non-carriers lost 6.1±0.2% of their initial weight. Therefore, allele carriers can lose similar weight during calorie restriction suggesting again that in the absence of environmental factors MC4R variants do not affect the ability to lose weight. While MC4R rare variants are highly associated with obesity, external factors contribute significantly to the obese phenotype. At the cellular level no consistent change in MC4R signaling can account for the association of rare variants with obesity. For example, only two of the rare mutations, I137T and C326R, had defects in cAMP signaling, while the other mutations signaled as well as wild-type MC4R. The defects of the I137T and C326R would only account for ∼25% reduction in function of one copy of MC4R. This small defect may contribute to the obesity of those carrying these variants; however, only the I137T carrier failed to lose similar weight compared to a matched subject post-RYGB. While there may be a common unifying mechanism for all the obesity associated MC4R variants, none has been reported to date.A wealth of data exist that show association between carrying rare, presumably deleterious, Together these data highlight the complex nature of MC4R signaling and how it affects not only obesity and metabolism, but also weight-loss. Altered MC4R function does not rule out the effect of other factors that may impact both obesity and RYGB outcomes. We suggest that increasing MC4R function will have a positive outcome on weight-loss after caloric restriction or RYGB, as well as improved T2D remission after RYGB. While several MC4R agonists have failed in clinical trials, mainly due to unrelated side effects, they do hold significant promise for weight-loss, which may depend on concomitant modification in feeding behavior.Figure S150 value for WT-MC4R is 41.1 nM and for G34A is 43.0 nM which were not statistically different (ANOVA with Dunnet's post-hoc test).cAMP dose-response of MC4R and variant G34A. cAMP dose-response in HEK cells stably expressing HA-MC4R (○ solid black lines) and the mutant G34A (□ dashed line) from three independent immunoassays. The α-MSH EC(TIF)Click here for additional data file.Methods S1Supplemental Methods.(DOCX)Click here for additional data file.