Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: Deep sequencing of the ST18 gene locus in 16 PV patients led to identification of 789 genetic variants (black dots), depicted along the ST18 gene locus (X axis, chr8:53,023,392–53,373,519, GRCh37/hg19 assembly; Y axis, negative log-transformed P-values of association score). A case-control association analysis against the 1000 genome project data revealed a large PV-associated haplotype block (in red) residing within an ST18 intron and harboring rs17315309 (arrows).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Sequencing, Transformation Assay, Control, Blocking Assay

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: (a) rs17315309 resides within a p53/p63 half binding site, upstream to the ST18 coding sequence, and results in the substitution of a highly conserved T nucleotide (broken arrow) within the consensus binding sites of p53 and p63 (MotifMap database; http://motifmap.ics.uci.edu/ ); (b) NHEKs were co-transfected with a luciferase reporter construct under the regulation of a 282 bp fragment from the putative ST18 promoter, harboring either the wild type or the risk allele of rs17315309, and with control (si-Cont), TP53- (si-p53) or TP63- (si-p63) specific siRNAs. Luciferase activity measurements are provided as arbitrary units (a.u.) relative to the luciferase activity measured in cells transfected with wild type allele and control siRNA. Results represent the mean of three independent experiments ± SE (*p<0.05, **p<0.01 and ***p<0.001 by 2-tailed t test).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Construct, Control, Activity Assay

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: NHEKs were transfected with an ST18 expression vector (ST18, dark grey) or control empty vector (EV, light grey). (a,b,c) Supernatants were collected 2, 4, 6 and 24 hours post exposure to PV serum or control serum and (a) TNFα, (b) IL-1α and (c) IL-6 secretion was measured as described in Materials and Methods; (d,e,f) Supernatants were collected 6 hours (IL-1α and IL-6) or 24 hours (TNFα) post exposure to PV and control serum or PV and control IgG and (d) TNFα, (e) IL-1α and (f) IL-6 secretion was measured as described in Materials and Methods. Results represent the mean of three independent experiments and are expressed as the relative cytokine secretion in percentage, compared to control (empty vector) ± SE (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by 2-tailed t test).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Transfection, Expressing, Plasmid Preparation, Control

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: We used a dispase-based dissociation assay to evaluate the effect of ST18 overexpression on cell adhesion. NHEKs transfected with a ST18 expression vector (ST18) or with a control vector (EV) were grown to confluency in the presence of PV serum and control serum or PV IgG and control IgG. (a) Epidermal sheets were released from the tissue plates and subjected to mechanical stress as described in Materials and Methods; (b) the resulting fragments were counted. Results represent the mean of three independent experiments and are expressed as number of fragments ± SE (*p<0.05, **p<0.01, ***p<0.001 by 2-tailed t test).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Control

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: The expression cassette for C-terminal ( a ) and N-terminal ( b ) anchoring of CCL3Gag in L. plantarum. All parts of the cassette are easily exchangeable using restriction sites: SalI between the signal peptide or the N-terminal anchor and CCL3Gag, EcoRI , at the end of the insert, and MluI between CCL3Gag and the C-terminal anchor. a C-terminal anchoring was accomplished by fusing the N-terminus of the CCL3Gag fragment to a signal peptide (SP; from Lp_0373) and the C-terminus to a cell wall anchor (Cwa) from Lp_2578 (Cwa comprises 194 residues of Lp_2578) . b Four N-terminal anchoring motifs were used, all containing an SP. Two lipoanchors were generated using lipobox fragments from Lp_1261 (residues 1–75) and Lp_1452 (residues 1–142), one transmembrane anchor was generated by fusing CCL3Gag to C-terminally truncated Lp_1568 (complete protein with 7-residues truncation), which contains an SP without a predicted signal peptide cleavage site, and one LysM anchor was generated by fusing CCL3Gag to full length Lp_3014 (a 204 residue putative transglycosylase with an N-terminal LysM domain)

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Expressing, Generated, Residue

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: Detection of CCL3Gag fusion protein produced in L. plantarum strains harboring various plasmids. Bacterial cell-free protein extracts were prepared 3 h after induction by use of peptide pheromone, and analyzed by Western blotting, using polyclonal goat anti-CCL3 antibody and polyclonal rabbit anti-goat HRP-conjugated IgG. A strain harboring the pEV plasmid , not containing the ccl3gag fragment, was used as a negative control. The following CCL3Gag producing strains were analyzed (expected mass of the CCL3Gag protein between parenthesis): Lp _1452CCL3Gag (39.9 kDa), Lp _1568CCL3Gag (95.3 kDa), Lp _3014CCL3Gag (45.0 kDa), Lp _1261CCL3Gag (31.9 kDa), Lp _CCL3Gag_Cwa (50.5 kDa). The arrows indicate the location of the fusion proteins. Lane M shows a molecular mass standard

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Produced, Western Blot, Plasmid Preparation, Negative Control

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: Growth of CCL3Gag-expressing L. plantarum cells. OD 600 values were measured at the induction point ( black bars ). Next, each culture was divided into a pheromone induced culture ( white bars ) and a non-induced culture ( gray bars ). OD 600 values were measured 3 h after the point of induction. The data are presented as the means from triplicates +SD

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Expressing

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: Flow cytometry ( a ) and microscopy ( b ) analysis of surface localization of CCL3Gag . L. plantarum cells harboring plasmids designed for N- or C-terminal anchoring of CCL3Gag were probed with goat anti-CCL3 polyclonal antibody and, subsequently, Alexa Fluor ® 488-conjugated rabbit anti-goat IgG antibodies. L. plantarum harboring pEV without the ccl3gag gene fragment was used as a negative control and is shown in all five histograms in panel a ( black line ). The data are presented as one representative experiment. Each experiment was performed at least three independent times and gave the similar results

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Flow Cytometry, Microscopy, Negative Control

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: Chemotactic properties of L. plantarum harbouring various constructs. The graphs show migration of Esb-MP cells towards CCL3Gag-expressing strains compared to migration towards the negative control ( Lp _Ev). The number of migrated cells was counted using flow cytometry and relative chemotaxis is shown as the average fold change relative to the negative control. a L. plantarum strain displaying CCL3Gag anchored C-terminally to the surface. The data presented are the means from 3 replicates +SEM. Statistically significant differences compared to the negative control ( p < 0.01) are indicated by an asterisk (*). b L. plantarum strains displaying CCL3Gag anchored N-terminally to the surface. The data presented are derived from one representative experiment. The experiments were performed at least three times and these independent experiments showed the similar trends

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Construct, Migration, Expressing, Negative Control, Flow Cytometry, Chemotaxis Assay, Derivative Assay

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: Chemotactic properties of L. plantarum strains surface displaying CCL3Gag, in presence of soluble CCL3 protein. Migration of Esb-MP cells towards CCL3Gag expressing strains in the presence of 3 ng/ml free CCL3 was compared to migration towards the negative control ( Lp _Ev). The number of migrated cells was counted using flow cytometry and relative chemotaxis is shown as average fold change relative to the negative control. The data are presented as the means from at least 5 replicates +SEM. Statistically significant differences ( p < 0.01) are indicated by an asterisk (*)

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Migration, Expressing, Negative Control, Flow Cytometry, Chemotaxis Assay

Journal: Microbial Cell Factories

Article Title: Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

doi: 10.1186/s12934-015-0360-z

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: The ccl3gag gene fragment was codon optimized for expression in L. plantarum, synthesized at Genscript (Piscataway, NJ) and cloned into a pUC57 plasmid, yielding pUC-CCL3Gag.

Techniques: Plasmid Preparation, Control, Negative Control