Journal: Archives of insect biochemistry and physiology

Article Title: Identification of the western tarnished plant bug (Lygus hesperus) olfactory co-receptor Orco: expression profile and confirmation of atypical membrane topology.

doi: 10.1002/arch.21042

Figure Lengend Snippet: Figure 6. Localization and topological orientation of transiently expressed LhOrco in cultured Tni cells. (A) Expression of LhOrco-EGFP fluorescent chimeras. Chimeras were generated by OE-PCR with EGFP at the N terminus (EGFP-LhOrco) or C terminus (LhOrco-EGFP). No cell surface localization was observed in either construct. (B) Fluorescent immunohistochemistry of 6x-His tagged LhOrco in the absence of cell permeabi- lization. Fixed Tni cells transfected with plasmids encoding LhGαi-His (C-terminal 6x-His tag), His-LhOrco (N-terminal 6x-His tag), LhOrco-His (C-terminal 6x-His tag), or mock transfected (Tni alone) were probed with a polyclonal mouse anti-His antibody (primary) and a goat anti-mouse IgG-TRITC antibody (secondary). In the absence of cell permeabilization, fluorescence was only observed in cells expressing LhOrco-His, indi- cating an extracellular C terminus. (C) Fluorescent immunohistochemistry of 6x-His tagged LhOrco following cell permeabilization. Cells are as in (B). In the presence of cell permeabilization, fluorescence is seen in the His-LhOrco cells, indicating an intracellular N terminus. Fluorescence, as expected, was also observed in the LhGαi-His expressing cells. Scale bar represents 20 μm. Images are representative of multiple independent transfections.

Article Snippet: Cells were then incubated for 2 h at 25◦C with a rabbit polyclonal anti-His antibody (SC-804, 200 μg/mL; Santa Cruz Biotech.

Techniques: Cell Culture, Expressing, Generated, Overlap Extension Polymerase Chain Reaction, Construct, Immunohistochemistry, Transfection, Fluorescence

Journal: Infection and Immunity

Article Title: Characterization of Heat-Stable (STa) Toxoids of Enterotoxigenic Escherichia coli Fused to Double Mutant Heat-Labile Toxin Peptide in Inducing Neutralizing Anti-STa Antibodies

doi: 10.1128/IAI.01394-13

Figure Lengend Snippet: Construction and detection of STa-toxoid-dmLT toxoid fusions. (A) Illustration of an STa-toxoid-dmLT toxoid fusion gene. Three copies of each STa-toxoid gene were genetically fused to the 5′ end, between A1 and A2 of LTA, and the 3′ end of a monomeric dmLT peptide gene (LTR192G/L211A; as a single open reading frame) using splicing overlap extension PCRs. Primers: 1, T7-F; 2, STa-toxoid-F; 3, STa-toxoid-R; 4, LT73-R; 5, LT73-F; 6, LT211-R; 7, LT211-F; 8, T7-R. Primers 2 and 3 were used to mutate the STa gene for different STa toxoids (Table 2). Primers 1 and 3 and primers 2 and 4 were, respectively, used in two PCRs (which generated two nucleotide fragments to be fused) to produce the first segment of the fusion gene, which included the first copy of a STa toxoid and the first 75 amino acids of the LT A subunit (at the N terminus). Primers 5 and 3 and primers 2 and 6 were used in two other PCRs (for two other fragments to be fused) to generate the second segment of the fusion gene, which consisted of the second copy of a STa toxoid and amino acids 68 to 216 of the LT A subunit. Primers 7 and 3 and primers 2 and 8 were used in another two PCRs (for another two fragments to be fused) to create the third segment of the fusion gene, which contains the third copy of a STa toxoid, amino acids 204 to 240 of the LT A subunit and the LT B subunit (1 to 100 amino acids). These three segments were connected in an SOE PCR for a single open reading frame coding a STa-toxoid-dmLT toxoid fusion protein. (B) Western blot to detect each STa-toxoid-dmLT fusion protein with anti-CT antibodies. A fusion protein (100 ng) separated in 10 to 12% PAGE gel was detected using rabbit anti-CT antiserum (1:3,300; Sigma) and IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR). (C) Western blot to detect toxoid fusion proteins with anti-STa antibodies. Fusion proteins (100 ng each) were detected with protein A-purified rabbit anti-STa antiserum (1:5,000) and IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR). The total protein extracted from host strain 8955 was used as the negative control (−). Lane M is the protein marker (in kilodaltons; Precision Plus Protein Pre-stained standards; Bio-Rad).

Article Snippet: IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR, Lincoln, NE) were used as the secondary antibody to detect each fusion protein with a LI-COR Odyssey premium infrared gel imaging system.

Techniques: Generated, Overlap Extension Polymerase Chain Reaction, Western Blot, Labeling, Purification, Negative Control, Marker, Staining

Journal: Antioxidants

Article Title: Nephroprotective Effects of Synthetic Flavonoid Hidrosmin in Experimental Diabetic Nephropathy

doi: 10.3390/antiox10121920

Figure Lengend Snippet: Hidrosmin effects on senescence markers in diabetic kidneys: ( a ) real-time PCR analysis of the cell cycle inhibitor p16 INK4a gene expression in renal samples. Values normalized to 18S gene are expressed as arbitrary units (a.u.); ( b , c ) representative images (magnification ×400) and quantification of p16 INK4a protein ( b ) and DNA damage biomarker p-H2A.X ( c ) in kidney sections from of untreated (D) and hidrosmin-treated (D + H) diabetic mice. The graphs represent individual values and the mean ± SEM of n = 9 animals (D group) and n = 11 animals (D + H group). ** p < 0.01 and *** p < 0.001 vs. D.

Article Snippet: After antigen retrieval (0.01 M citrate buffer pH 6 for 20 min) and blockade of endogenous peroxidase (3% H 2 O 2 in methanol for 30 min) and nonspecific binding (8% host serum for 30 min), slides were incubated overnight at 4 °C with primary antibodies against CD3 (Agilent Cat# A0452, RRID:AB_2335677; Santa Clara, CA, USA), F4/80 (Bio-Rad Cat# MCA497R, RRID:AB_323279; Hercules, CA, USA), phosphorylated (p-)STAT3 (Cell Signaling Technology Cat# 9134, RRID:AB_331589; Danvers, MA, USA), p-p65 (Santa Cruz Biotechnology Cat# sc-136548, RRID:AB_10610391; Santa Cruz, CA, USA), p-NRF2 (Abcam Cat# ab76026, RRID:AB_1524049), p16 INK4a (Thermo Fisher Scientific Cat# MA5-17142, RRID:AB_2538613; Waltham, MA, USA), and p-H2A histone family member X (H2A.X)(Cell Signaling Technology Cat# 9718, RRID:AB_2118009).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Biomarker Assay

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: (a) Amplification of the VL and VH genes from immunized mice and screening of phage display scFv antibodies generated from the third round of biopanning. Lane M: DNA Marker 100 bp (Fermentas); lane VL: PCR products of the VL gene; lane VH: PCR products of the VH gene; lane VL–VH: VL gene joined with VH gene by SOE-PCR, and (b) screening of high affinity phage display scFv antibody to the N-protein using an indirect ELISA.

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Amplification, Generated, Marker, Overlap Extension Polymerase Chain Reaction, Indirect ELISA

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: Screening of a scFv antibody pair sensitive and specific to the N-protein of SARS-CoV-2. (a) A high-affinity pair of capture scFv and detection scFv conjugated with HRP to the N-protein of SARS-CoV-2 was screened by a sandwich ELISA, and (b) specificity of capture antibody #4 and detection antibody #2 was examined by a sandwich ELISA using the N-protein of SARS-CoV-2.

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Sandwich ELISA

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: Extraction scheme of N-protein from pseudovirus present in a nasopharyngeal swab sample using an extraction buffer.

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Extraction

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: Standard curve obtained for assay on the Optimiser microplate for N-protein extracted from pseudovirus, detected using the custom designed scFv antibody pair and compared with the commercially available IgG antibody pair from Sino Biological. Each data point is the mean of triplicate, and the error bar represents the standard deviation. The LoD of the developed scFv antibody pair was 10 3 copies per sample, while the LoD of the Sino Biological antibody pair was 10 5 copies per sample.

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Standard Deviation

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: Standard curve obtained for assay on Optimiser™ microplate for N-protein in spiked sample. Each data point is the mean of triplicate and error bar represents the standard deviation. LoD was measured as 1.6 ng mL −1 .

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Standard Deviation

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: Performance comparison of the mLFA-LOC platform with the commercial RDT. (a) Standard curve obtained for the assay with N-protein extracted from pseudovirus on mLFA, quantified using portable reader and compared with BioTek reader. Each data point is the mean of triplicate, and the error bar represents the standard deviation, and (b) assay performed with N-protein extracted from pseudovirus on commercial RDT as per the kit’s instructions (Flowflex®, USA). LoD of the CL-mLFA on LOC was 10 3 copies per sample whereas the LoD of the commercial RDT was 10 5 copies per sample.

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Comparison, Standard Deviation

Journal: Sensors & Diagnostics

Article Title: A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

doi: 10.1039/d4sd00352g

Figure Lengend Snippet: Standard curve obtained for N-protein assay with spiked sample on mLFA, quantified using the portable reader and compared with BioTek reader. Each data point is the mean of triplicate, and the error bar represents the standard deviation. LoD was measured as 1.6 ng mL −1 .

Article Snippet: Two 6 week-old Balb/c mice (Jackson Laboratories, USA) were immunized via a footpad injection with a final volume of 15 μL containing 25 μg of recombinant N-protein of SARS-CoV-2 ( YP_009724397.2 , Sino Biological, China), emulsified in an equal volume of TiterMax adjuvant (Millipore-Sigma, USA).

Techniques: Standard Deviation