Journal: Microbiology Spectrum

Article Title: Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

doi: 10.1128/Spectrum.02094-21

Figure Lengend Snippet: Summary of affinity selection results against Bradyrhizobium strains SUTN9-2 and DOA9

Article Snippet: For larger scale production of soluble scFv antibodies, the gene encoding for scFv antibody clones: yiN92-1e10 and yiDOA9-162 were digested with NcoI and NotI restriction enzymes and sub-cloned into pET-21d (+) vector (NEB, USA), to generate pET21d+/yiN92-1e10 and pET21d+/yiDOA9-162 plasmids.

Techniques: Selection, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: Microbiology Spectrum

Article Title: Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

doi: 10.1128/Spectrum.02094-21

Figure Lengend Snippet: Specific binding of selected phage-displayed scFv clones. Phage ELISA result of the binding of scFv antibody clones yiDOA9-162 and yiN92-1e10 against strain DOA9 and SUTN9-2, respectively in pure culture (boiled and nonboiled cells). The average OD 405 nm values and standard errors from triplicated wells are shown.

Article Snippet: For larger scale production of soluble scFv antibodies, the gene encoding for scFv antibody clones: yiN92-1e10 and yiDOA9-162 were digested with NcoI and NotI restriction enzymes and sub-cloned into pET-21d (+) vector (NEB, USA), to generate pET21d+/yiN92-1e10 and pET21d+/yiDOA9-162 plasmids.

Techniques: Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: Microbiology Spectrum

Article Title: Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

doi: 10.1128/Spectrum.02094-21

Figure Lengend Snippet: Binding property of free soluble scFv antibodies. ELISA results of soluble scFv antibodies (yiN92-1e10 and yiDOA9-162) against each target strain in planktonic form (A) and bacteroid form inside the nodules of Bradyrhizobium DOA9 and SUTN9-2 obtained from A . americana and V . radiata , respectively (B). Values are the mean of triplicate wells. Error bars show the standard deviation for each set of data.

Article Snippet: For larger scale production of soluble scFv antibodies, the gene encoding for scFv antibody clones: yiN92-1e10 and yiDOA9-162 were digested with NcoI and NotI restriction enzymes and sub-cloned into pET-21d (+) vector (NEB, USA), to generate pET21d+/yiN92-1e10 and pET21d+/yiDOA9-162 plasmids.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

doi: 10.1128/Spectrum.02094-21

Figure Lengend Snippet: Confocal laser scanning micrographs of Bradyrhizobium strains SUTN9-2 and DOA9 prepared from pure culture and plant nodules. The bacterial samples were stained with two different scFv antibodies: yiN92-1e10 and yiDOA9-162. Green spot indicated the green-fluorescent staining of scFv antibody, using secondary antibody conjugated to anti-His-Dylight 488. Plant cell walls were stained with a blue fluorophore (Calcofluor white M2R) and emitted blue color. The bacteroides are shown as green spots inside a blue plant cavity. The nodule images observed by differential interference contrast (DIC) technique were also indicated. Scale bar is 10 μm at ×600 magnification, and 100 μm at ×100 magnification for pure culture and nodule samples, respectively.

Article Snippet: For larger scale production of soluble scFv antibodies, the gene encoding for scFv antibody clones: yiN92-1e10 and yiDOA9-162 were digested with NcoI and NotI restriction enzymes and sub-cloned into pET-21d (+) vector (NEB, USA), to generate pET21d+/yiN92-1e10 and pET21d+/yiDOA9-162 plasmids.

Techniques: Staining

Journal: Microbiology Spectrum

Article Title: Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

doi: 10.1128/Spectrum.02094-21

Figure Lengend Snippet: Cross reactivity of scFv antibody by ELISA. Binding activities of yiN92-1e10 (A) and yiDOA9-162 (B) scFv were determined by ELISA on a plate coated with 5 μg of various target antigens as indicated in the graphs. Boiled bacterial targets for yiN92-1e10 scFv (A) are Bradyrhizobium strains, SUTN9-2, PRC008, USDA110, DOA9, DOA1, ORS3257, 194, CB1809, S23321, ORS278, TAL173, DASA02002, DASA02082, DASA02042, DASA02068, DASA02193, DASA02198, peanut bradyrhizobia isolates no. 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13 and 1% BSA. Boiled bacterial targets for yiDOA9-162 scFv (B) are Bradyrhizobium strains DOA9, DOA4, DOA1, SUTN1-12, SUTN9-2, SUTN7-2, B. diazoefficiens USDA110, other rhizobia bacteria ( Sinorhizobium sp. BL3, Azorhizobium sp., Allorhizobium sp., Mesorhizobium loti MAFF), Erwinia sp. (pathogenic bacterium), and 1% BSA. The wells were detected with 5 μg of scFv antibody. One percent BSA was served as negative control. Values are the mean of triplicate wells. Error bars show the standard deviation for each set of data.

Article Snippet: For larger scale production of soluble scFv antibodies, the gene encoding for scFv antibody clones: yiN92-1e10 and yiDOA9-162 were digested with NcoI and NotI restriction enzymes and sub-cloned into pET-21d (+) vector (NEB, USA), to generate pET21d+/yiN92-1e10 and pET21d+/yiDOA9-162 plasmids.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

doi: 10.1128/Spectrum.02094-21

Figure Lengend Snippet: Persistence and nodulation of Bradyrhizobium SUTN9-2 from rice stubbles in mung bean. ELISA of bacterial cells from soil suspension (A) and bacteroid from nodule (B). The values are the mean of triplicate wells. Error bars show the standard deviation for each set of data. (C) Confocal laser scanning micrographs of nodule inoculated with SUTN9-2. The cross sections of nodule were stained with scFv yiN92-1e10 and yiDOA9-162. Green spot indicated the green-fluorescent staining of scFv antibody, using secondary antibody conjugated with anti-His Dylight 488. Plant cell walls were stained with a blue fluorophore (Calcofluor white M2R) and emitted blue color. The bacteroides are shown as green spots inside a blue plant cavity. The nodule images observed by differential interference contrast (DIC) technique were also indicated. Scale bar is 100 μm at ×100 magnification, and 50 μm at ×400 magnification.

Article Snippet: For larger scale production of soluble scFv antibodies, the gene encoding for scFv antibody clones: yiN92-1e10 and yiDOA9-162 were digested with NcoI and NotI restriction enzymes and sub-cloned into pET-21d (+) vector (NEB, USA), to generate pET21d+/yiN92-1e10 and pET21d+/yiDOA9-162 plasmids.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Staining

Journal: PLoS Genetics

Article Title: The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A

doi: 10.1371/journal.pgen.1006665

Figure Lengend Snippet: (A) Female and male Coucou de Rennes chicken with separately depicted feather illustrating the iconic Sex-linked barring phenotype caused by the B1 allele. (B) Sex-linked barring alleles and associated sequence variants. SNP1 and SNP2 are non-coding while SNP3 and SNP4 constitute non-synonymous changes in the region encoding the MDM2 binding domain. (C) Sex-linked dilution phenotype caused by the B2 allele. Note how the homozygous male has an almost white appearance whereas the hemizygous female as well as the heterozygous male show a Sex-linked barring pattern. (D) Phenotype with individual feathers from N/N , B2/N and B0/N chicken. Photo credits: (A) Hervé Ronné, Ecomusée du pays de Rennes, (C) Susanne Kerje, (D) Dominic Wright and Doreen Schwochow-Thalmann.

Article Snippet: A truncated version of chicken MDM2 (NCBI accession number NP_001186313) containing amino acid residues 204–298 (MDM2 204-298 ) was synthesized (GenScript) and cloned into the expression vector pSY5, a modified pET-21d(+) plasmid (Novagen), encoding an 8-histidine tag ahead of the N-terminus of the protein [ ].

Techniques: Sequencing, Binding Assay

Journal: PLoS Genetics

Article Title: The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A

doi: 10.1371/journal.pgen.1006665

Figure Lengend Snippet: (A) Far-UV circular dichroism (CD) is measured in mDeg and monitors protein secondary structure. Spectra of MDM2 204-298 , ARF 1-14 WT , and complexes of ARF 1-14 WT /MDM2 204-298 , ARF 1-14 V9D /MDM2 204-298 , and ARF 1-14 R10C /MDM2 204-298 . MDM2 204-298 was used at 10 μM and all peptides at 64 μM final concentrations. Spectra of all peptides in free form and at 25 μM concentration in complex with MDM2 204-298 are shown in . (B) Isothermal titration calorimetry (ITC) experiments in which the heat (enthalpy) associated with binding is recorded as μcal/sec for each titration point (top panel) and integrated and normalized against the concentrations of ARF peptide and MDM2 204-298 to obtain a binding isotherm (expressed as kcal mol -1 versus molar ratio; bottom panel). In the experiment, ARF peptides (WT, V9D, and R10C, respectively) were titrated into 100 μM MDM2 204-298 with the peptide concentration increasing by approximately 10 μM in each titration point. Peaks (top) and integrated energies (bottom) corrected for heats of dilution are shown. Raw data are shown in . (C) Assessment of the effect of the two coding mutations on the interaction between ARF and MDM2 based on a luciferase assay. Reduced luciferase activity implies weaker interaction between ARF and MDM2, monitored as a decreased ability of ARF to protect the transcription factor p53 from degradation (Student’s t-test; * P <0.05, *** P <0.001).

Article Snippet: A truncated version of chicken MDM2 (NCBI accession number NP_001186313) containing amino acid residues 204–298 (MDM2 204-298 ) was synthesized (GenScript) and cloned into the expression vector pSY5, a modified pET-21d(+) plasmid (Novagen), encoding an 8-histidine tag ahead of the N-terminus of the protein [ ].

Techniques: Circular Dichroism, Concentration Assay, Isothermal Titration Calorimetry, Binding Assay, Titration, Luciferase, Activity Assay

Journal: PLoS Genetics

Article Title: The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A

doi: 10.1371/journal.pgen.1006665

Figure Lengend Snippet: (A) The non-coding mutation(s) present in the B0 , B1 and B2 allele cause a tissue specific up-regulation of CDKN2A encoding the ARF protein. ARF inhibits MDM2-mediated degradation of p53. p53 will activate downstream targets possibly initiating premature melanocyte differentiation and thereby loss of mature pigment cells. (B) The missense mutations present in the B1 and B2 alleles impair the interaction between ARF and MDM2, which counteract the consequences of up-regulated ARF expression. (C) In solid colored feathers, melanocyte progenitor cells migrate up from the feather base and start expressing CDKN2A in the barb region leading to differentiation of melanocytes and pigment production without exhausting the pool of undifferentiated melanocytes. In sex-linked barred feathers, up-regulated ARF expression may lead to premature differentiation of pigment cells and a lack of undifferentiated melanocytes that can replenish the ones producing pigment. As the feather keeps on growing, no more melanocytes are available to produce pigment resulting in the white bar. A plausible explanation for the cyclic appearance of white and black bars is that new recruitment of melanocyte progenitor cells takes place after the undifferentiated melanocytes have been depleted.

Article Snippet: A truncated version of chicken MDM2 (NCBI accession number NP_001186313) containing amino acid residues 204–298 (MDM2 204-298 ) was synthesized (GenScript) and cloned into the expression vector pSY5, a modified pET-21d(+) plasmid (Novagen), encoding an 8-histidine tag ahead of the N-terminus of the protein [ ].

Techniques: Mutagenesis, Expressing

Journal: Life Science Alliance

Article Title: An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65

doi: 10.26508/lsa.202201546

Figure Lengend Snippet: (A) Three-dimensional structure of bovine RPE65 (PDB ID: 3FSN ). The crystallographically unresolved (aa107–125) region of RPE65 protein, modeled using I-TASSER server, is shown in red color. (B) Alignment of RPE65 aa107–125 from several jawed and jawless vertebrate taxa showing sequence conservation throughout evolution. (C) Helical wheel representation of the crystallographically unresolved aa107–125 region of RPE65 protein using HELIQUEST server. (D) Predicted secondary structure, relative accessibility and hydropathy analysis of the aa107–125 region of RPE65 protein using I-TASSER and ENDScript server, respectively. (E) Hydropathic moment plot of combined 35 different RPE65 aa107–125 and paralogous conserved region sequences from vertebrate and non-vertebrate carotenoid cleavage dioxygenase (CCD) paralogs, derived from HeliQuest . Red stars, RPE65 s; magenta circles, vertebrate BCO1s; blue circles, vertebrate BCO2s; black circle, early chordate ( Ciona ) CCD; yellow circles, arthropod ( Trinorchestia , Palaemon , Drosophila , and Galleria ) CCDs; and green circles, nematode ( Caenorhabditis ) -CCDs. Note tight grouping of all 10 RPE65 s distinct from other paralogs. (F) Physicochemical properties of the crystallographically unresolved (aa107–125) of RPE65 protein. Abbreviations: acc, accessibility; hyd, hydrophobicity.

Article Snippet: We cloned the crystallographically unresolved region (107–125 aa, AH 107-125 ) of RPE65 protein into the MBP fusion cloning vector pET His6 MBP TEV LIC (2M-T; a gift from Scott Gradia, plasmid # 29708; Addgene).

Techniques: Sequencing, Derivative Assay