Journal: Applied Microbiology and Biotechnology

Article Title: A broadly applicable split-luciferase biosensor approach for rapid antibody detection in emerging infectious diseases

doi: 10.1007/s00253-026-13712-5

Figure Lengend Snippet: Sensor pair selection for detection of SARS-CoV-2 and CHIKV antibodies. a Different sensor combinations were screened for their ability to reconstitute luciferase activity in the presence of anti-SARS-CoV-2 RBD antibodies. b Sensor pairs were similarly evaluated for luciferase complementation in the presence of anti-CHIKV E2 antibodies. Data represent means of duplicate tests; error bars indicate standard deviations

Article Snippet: Plates were coated with SARS-CoV-2 spike RBD ( P08128 , GenBank accession no. NC_045512 ; Solarbio, China) or CHIKV E2 (strain SL-CK1; 40,440-V08B; GenBank accession no. ADG95913 ; Sino Biological, China) at 0.1 μg/well at 4 °C overnight, washed with PBST, and blocked with 2% BSA for 1 h at 37 °C.

Techniques: Selection, Luciferase, Activity Assay

Journal: Applied Microbiology and Biotechnology

Article Title: A broadly applicable split-luciferase biosensor approach for rapid antibody detection in emerging infectious diseases

doi: 10.1007/s00253-026-13712-5

Figure Lengend Snippet: Analytical performance of the bioluminescent immunoassay for detection of anti-SARS-CoV-2 RBD (6.39 mg/mL) and anti-CHIKV E2 antibodies (1.0 mg/mL). Data represent means of duplicate tests; error bars indicate standard deviations

Article Snippet: Plates were coated with SARS-CoV-2 spike RBD ( P08128 , GenBank accession no. NC_045512 ; Solarbio, China) or CHIKV E2 (strain SL-CK1; 40,440-V08B; GenBank accession no. ADG95913 ; Sino Biological, China) at 0.1 μg/well at 4 °C overnight, washed with PBST, and blocked with 2% BSA for 1 h at 37 °C.

Techniques:

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: Effect of RRM2 knockdown on DENV RNA replication and infectious virus production (A) Schematic representation of the experimental protocol. Briefly, HuH-7, HepG2, and A549 cells were transfected with RRM2 siRNA or non-targeting control siRNA (Ctrl.si) one day before infection with DENV-1 and DENV-2 at a MOI of 0.1. Cells and culture supernatants were harvested 72 h later to measure intracellular and extracellular DENV RNA levels using RT-qPCR and quantify infectious virus titers in culture supernatants via a focus-forming assay. Created using Biorender.com. (B) SDS-PAGE and western blotting were utilized to assess the efficiency of RRM2 knockdown in HepG2, HuH-7, and A549 cells after treatment with specific RRM2 siRNA for 96 h at final concentrations of 10, 20, 5, and 5 nM, respectively. The expression levels of other RR subunits, RRM1 and p53R2, were also characterized. β-actin served as a loading control. (C–E) Intracellular and extracellular DENV RNA copy numbers were measured 72 h post-transfection (hpi) via RT-qPCR in HuH-7, HepG2, and A549 cells. Intracellular DENV RNA levels were normalized to GAPDH mRNA levels. Results are presented as mean+/-standard deviation (SD). p values were assessed using Student’s t test. (F–H) Infectious virus titers in culture supernatants from non-transfected and siRNA-transfected HuH-7, HepG2, and A549 cells were determined using a fluorescent focus assay on BHK-21 cells. Results are presented as mean +/- SD. p values were evaluated by Student’s t test. (I) Viability of HuH-7, HepG2, and A549 cells following treatment with RRM2 siRNA. Cells were seeded in 96-well plates and transfected with the indicated final concentrations of RRM2 siRNA. At 96 h post-transfection, cell viability was assessed using the 2(2-methyl-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4disulfophenyl)-2H-tetrazolium, monosodium salt (WST)-8 assay (OD 450 ). The percentage of viability was calculated and compared to the untreated control (100% viability). (J) Immunofluorescence microscopy was conducted to evaluate the infection efficiency of DENV particles released from RRM2 or control siRNA-transfected and non-treated cells. Schematic representation of the experimental procedure. Culture supernatants harvested from non-transfected and siRNA-transfected DENV-infected HuH-7 and HepG2 cells were used to infect naive cell lines at an MOI of 1 (left). Three days post-infection, cell monolayers were analyzed by immunofluorescence for DENV envelope (E) protein expression using anti-flavivirus monoclonal antibody 4G2, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Representative immunofluorescence images (400× magnification) depicting DENV-E protein expression in HuH-7 and HepG2 cells. Cell nuclei were stained with DAPI, and cells were observed under a BZ-X700 fluorescence microscope (Keyence Co., Osaka, Japan). White squares indicate enlarged insets. Scale bars, 50 μm (right).

Article Snippet: Antibodies against the following proteins were used in the immunoblots: mouse anti-RRM2 clone 1E1 (cat# WH0006241M1; Sigma-Aldrich), rabbit anti-RRM2 (cat# GTX103193; GeneTex, Irvine, CA, USA), rabbit anti-furin (cat# PA1-062, Thermo Fisher Scientific), rabbit anti-RRM1 (cat# ab137114; Abcam, Cambridge, MA, USA), goat anti-p53R2 ( N -16, sc-10840, Santa Cruz Biotechnology, CA, USA), mouse anti-flavivirus envelope protein monoclonal antibody (clone 4G2; The Native Antigen Company, Oxford, UK), rabbit anti-dengue virus capsid protein (cat# GTX103343; GeneTex), rabbit anti-dengue virus prM protein (cat# GTX128093; GeneTex), mouse anti-dengue virus prM protein (clone CC5.A9.E10; The Native Antigen Company), mouse anti-dengue virus pr monoclonal antibody (cat# LDG0013YA; LEADGENE), mouse anti-dengue virus NS1 monoclonal antibody (cat# ab41490; Abcam), rabbit anti-mTOR (cat# GTX101557; GeneTex), rabbit anti-LC3I/II (cat# PM036; MBL International, Woburn, MA, USA), rabbit anti-4E-BP1 (cat# 9452; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-4E-BP1 (Ser65) (cat# 9451; Cell Signaling Technology), rabbit anti-phospho-4E-BP1 (Thr70) (cat# 9455; Cell Signaling Technology), and mouse anti-actin antibody (cat# A2228; Sigma-Aldrich).

Techniques: Knockdown, Virus, Transfection, Control, Infection, Quantitative RT-PCR, Focus Forming Assay, SDS Page, Western Blot, Expressing, Standard Deviation, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: RRM2 knockdown reduced furin protein levels in the cells, decreasing cleavage of DENV prM protein (A) RT-qPCR analysis of RRM2 mRNA levels in HuH-7 cells either mock-infected or infected with DENV-1 or DENV-2 at an MOI of 0.1. RRM2 mRNA levels were normalized to GAPDH mRNA levels. Data are expressed as mean ± standard deviation (SD) of triplicate measurements. p values were assessed using Student’s t test. n.s., not significant. (B) DENV infection increased RRM2 protein levels. HuH-7 cells were mock-infected or infected with DENV-1 or DENV-2 at an MOI of 0.1. Cells were harvested at the indicated time points (24, 48, and 72 hpi) and analyzed by western blotting using anti-RRM2, anti-p53R2, and mouse monoclonal anti-flavivirus E (4G2) antibodies. Endogenous β-actin expression served as an internal control. Protein levels were quantified using ImageJ software and normalized to β-actin levels. Densitometric p53R2/actin and RRM2/actin ratios are shown below the blots. (C) Representative western blot images depict RRM2, DENV envelope (E), DENV capsid, and DENV prM protein expression levels in HuH-7 cells that were transfected with RRM2 siRNA or control (Ctrl) siRNA (5 nM). HuH-7 cells were either non-transfected or transfected with siRNAs one day prior to infection with DENV-1 or DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and subjected to western blot analysis under non-reducing conditions using anti-prM, anti-capsid, anti-flavivirus E (4G2), and anti-RRM2 antibodies. β-actin was utilized as a loading control. (D) Determination of viral maturation was carried out by calculating the ratio of prM to E protein expression, normalized to non-transfected DENV-infected HuH-7 cells. Detected signals of DENV E and prM proteins in (C) were quantified using ImageJ software, and prM-to-E ratios are plotted as bar graphs. Numbers indicate n -fold increase compared to non-transfected cells. (E) Western blot analysis of prM and pr proteins in culture supernatants of non-transfected, control siRNA or RRM2 siRNA-transfected DENV-1 infected HuH-7 cells (C) under reducing conditions using an anti-pr mouse mAb. (F) Analysis of DENV E protein glycosylation status. Mock infected (−) or DENV (+) infected and siRNA-transfected HuH-7 cell lysates were treated with PNGaseF (+) or buffer control (−), then subjected to non-reducing SDS-PAGE and western blotting with anti-E 4G2, anti-RRM2, and anti-actin antibodies. Arrows indicate the positions of undigested and glycosylated E (2N) protein and deglycosylated forms (1N and 0N) of E protein. 0N, 1N, and 2N correspond to the number of N-linked glycans on E protein. β-actin was utilized as a loading control. (G) Quantification of endogenous furin mRNA levels relative to GAPDH in HuH-7 cells that were non-transfected or transfected with RRM2 siRNA or control siRNA (Ctrl si) (5 nM) for 96 h. Data are expressed as mean ± SD of triplicate measurements. (H) Effect of RRM2 knockdown on furin protein expression. HuH-7 cells were either non-transfected or transfected with control siRNA or RRM2 siRNA one day before mock infection or infection with DENV-1 or DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and analyzed by western blotting using anti-furin, anti-RRM2, and anti-DENV-E proteins. β-actin served as a loading control. Protein band intensities were analyzed using ImageJ software. Densitometric furin/actin ratios are shown below the blots. (I) Furin protein levels in HuH-7 and A549 cells with or without transfection of furin expression plasmid (myc-DDK-tagged human furin, 4 μg) and those non-transfected or transfected with control or RRM2 siRNA (5 nM). Densitometric furin/actin ratios are shown below the blots. (J) A549 cells were seeded in 60 mm dishes, and after 24 h, cells were transfected with the furin expression plasmid (4 μg) alone or co-transfected with RRM2 or control siRNA. The following day, the cells were infected with DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and subjected to western blot analysis under non-reducing conditions using anti-furin, anti-RRM2, anti-prM, anti-flavivirus E (4G2), and anti-actin antibodies. Densitometric prM/actin ratios are shown below the blots (left). Determination of viral maturation was carried out by calculating prM to E protein expression ratios normalized to those of control siRNA-transfected A549 cells. Detected signals of DENV E and prM proteins were quantified using ImageJ software as described in . prM-to-E ratios are plotted as bar graphs. Number indicates n -fold increase compared to Ctrl siRNA-transfected cells (right).

Article Snippet: Antibodies against the following proteins were used in the immunoblots: mouse anti-RRM2 clone 1E1 (cat# WH0006241M1; Sigma-Aldrich), rabbit anti-RRM2 (cat# GTX103193; GeneTex, Irvine, CA, USA), rabbit anti-furin (cat# PA1-062, Thermo Fisher Scientific), rabbit anti-RRM1 (cat# ab137114; Abcam, Cambridge, MA, USA), goat anti-p53R2 ( N -16, sc-10840, Santa Cruz Biotechnology, CA, USA), mouse anti-flavivirus envelope protein monoclonal antibody (clone 4G2; The Native Antigen Company, Oxford, UK), rabbit anti-dengue virus capsid protein (cat# GTX103343; GeneTex), rabbit anti-dengue virus prM protein (cat# GTX128093; GeneTex), mouse anti-dengue virus prM protein (clone CC5.A9.E10; The Native Antigen Company), mouse anti-dengue virus pr monoclonal antibody (cat# LDG0013YA; LEADGENE), mouse anti-dengue virus NS1 monoclonal antibody (cat# ab41490; Abcam), rabbit anti-mTOR (cat# GTX101557; GeneTex), rabbit anti-LC3I/II (cat# PM036; MBL International, Woburn, MA, USA), rabbit anti-4E-BP1 (cat# 9452; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-4E-BP1 (Ser65) (cat# 9451; Cell Signaling Technology), rabbit anti-phospho-4E-BP1 (Thr70) (cat# 9455; Cell Signaling Technology), and mouse anti-actin antibody (cat# A2228; Sigma-Aldrich).

Techniques: Knockdown, Quantitative RT-PCR, Infection, Standard Deviation, Western Blot, Expressing, Control, Software, Transfection, Glycoproteomics, SDS Page, Plasmid Preparation

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: Effect of RRM2 knockdown on DENV RNA replication and infectious virus production (A) Schematic representation of the experimental protocol. Briefly, HuH-7, HepG2, and A549 cells were transfected with RRM2 siRNA or non-targeting control siRNA (Ctrl.si) one day before infection with DENV-1 and DENV-2 at a MOI of 0.1. Cells and culture supernatants were harvested 72 h later to measure intracellular and extracellular DENV RNA levels using RT-qPCR and quantify infectious virus titers in culture supernatants via a focus-forming assay. Created using Biorender.com. (B) SDS-PAGE and western blotting were utilized to assess the efficiency of RRM2 knockdown in HepG2, HuH-7, and A549 cells after treatment with specific RRM2 siRNA for 96 h at final concentrations of 10, 20, 5, and 5 nM, respectively. The expression levels of other RR subunits, RRM1 and p53R2, were also characterized. β-actin served as a loading control. (C–E) Intracellular and extracellular DENV RNA copy numbers were measured 72 h post-transfection (hpi) via RT-qPCR in HuH-7, HepG2, and A549 cells. Intracellular DENV RNA levels were normalized to GAPDH mRNA levels. Results are presented as mean+/-standard deviation (SD). p values were assessed using Student’s t test. (F–H) Infectious virus titers in culture supernatants from non-transfected and siRNA-transfected HuH-7, HepG2, and A549 cells were determined using a fluorescent focus assay on BHK-21 cells. Results are presented as mean +/- SD. p values were evaluated by Student’s t test. (I) Viability of HuH-7, HepG2, and A549 cells following treatment with RRM2 siRNA. Cells were seeded in 96-well plates and transfected with the indicated final concentrations of RRM2 siRNA. At 96 h post-transfection, cell viability was assessed using the 2(2-methyl-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4disulfophenyl)-2H-tetrazolium, monosodium salt (WST)-8 assay (OD 450 ). The percentage of viability was calculated and compared to the untreated control (100% viability). (J) Immunofluorescence microscopy was conducted to evaluate the infection efficiency of DENV particles released from RRM2 or control siRNA-transfected and non-treated cells. Schematic representation of the experimental procedure. Culture supernatants harvested from non-transfected and siRNA-transfected DENV-infected HuH-7 and HepG2 cells were used to infect naive cell lines at an MOI of 1 (left). Three days post-infection, cell monolayers were analyzed by immunofluorescence for DENV envelope (E) protein expression using anti-flavivirus monoclonal antibody 4G2, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Representative immunofluorescence images (400× magnification) depicting DENV-E protein expression in HuH-7 and HepG2 cells. Cell nuclei were stained with DAPI, and cells were observed under a BZ-X700 fluorescence microscope (Keyence Co., Osaka, Japan). White squares indicate enlarged insets. Scale bars, 50 μm (right).

Article Snippet: Mouse anti-flavivirus envelope protein monoclonal antibody (D1-4G2-4-15) , ATCC , CVCL_J890.

Techniques: Knockdown, Virus, Transfection, Control, Infection, Quantitative RT-PCR, Focus Forming Assay, SDS Page, Western Blot, Expressing, Standard Deviation, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: RRM2 knockdown reduced furin protein levels in the cells, decreasing cleavage of DENV prM protein (A) RT-qPCR analysis of RRM2 mRNA levels in HuH-7 cells either mock-infected or infected with DENV-1 or DENV-2 at an MOI of 0.1. RRM2 mRNA levels were normalized to GAPDH mRNA levels. Data are expressed as mean ± standard deviation (SD) of triplicate measurements. p values were assessed using Student’s t test. n.s., not significant. (B) DENV infection increased RRM2 protein levels. HuH-7 cells were mock-infected or infected with DENV-1 or DENV-2 at an MOI of 0.1. Cells were harvested at the indicated time points (24, 48, and 72 hpi) and analyzed by western blotting using anti-RRM2, anti-p53R2, and mouse monoclonal anti-flavivirus E (4G2) antibodies. Endogenous β-actin expression served as an internal control. Protein levels were quantified using ImageJ software and normalized to β-actin levels. Densitometric p53R2/actin and RRM2/actin ratios are shown below the blots. (C) Representative western blot images depict RRM2, DENV envelope (E), DENV capsid, and DENV prM protein expression levels in HuH-7 cells that were transfected with RRM2 siRNA or control (Ctrl) siRNA (5 nM). HuH-7 cells were either non-transfected or transfected with siRNAs one day prior to infection with DENV-1 or DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and subjected to western blot analysis under non-reducing conditions using anti-prM, anti-capsid, anti-flavivirus E (4G2), and anti-RRM2 antibodies. β-actin was utilized as a loading control. (D) Determination of viral maturation was carried out by calculating the ratio of prM to E protein expression, normalized to non-transfected DENV-infected HuH-7 cells. Detected signals of DENV E and prM proteins in (C) were quantified using ImageJ software, and prM-to-E ratios are plotted as bar graphs. Numbers indicate n -fold increase compared to non-transfected cells. (E) Western blot analysis of prM and pr proteins in culture supernatants of non-transfected, control siRNA or RRM2 siRNA-transfected DENV-1 infected HuH-7 cells (C) under reducing conditions using an anti-pr mouse mAb. (F) Analysis of DENV E protein glycosylation status. Mock infected (−) or DENV (+) infected and siRNA-transfected HuH-7 cell lysates were treated with PNGaseF (+) or buffer control (−), then subjected to non-reducing SDS-PAGE and western blotting with anti-E 4G2, anti-RRM2, and anti-actin antibodies. Arrows indicate the positions of undigested and glycosylated E (2N) protein and deglycosylated forms (1N and 0N) of E protein. 0N, 1N, and 2N correspond to the number of N-linked glycans on E protein. β-actin was utilized as a loading control. (G) Quantification of endogenous furin mRNA levels relative to GAPDH in HuH-7 cells that were non-transfected or transfected with RRM2 siRNA or control siRNA (Ctrl si) (5 nM) for 96 h. Data are expressed as mean ± SD of triplicate measurements. (H) Effect of RRM2 knockdown on furin protein expression. HuH-7 cells were either non-transfected or transfected with control siRNA or RRM2 siRNA one day before mock infection or infection with DENV-1 or DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and analyzed by western blotting using anti-furin, anti-RRM2, and anti-DENV-E proteins. β-actin served as a loading control. Protein band intensities were analyzed using ImageJ software. Densitometric furin/actin ratios are shown below the blots. (I) Furin protein levels in HuH-7 and A549 cells with or without transfection of furin expression plasmid (myc-DDK-tagged human furin, 4 μg) and those non-transfected or transfected with control or RRM2 siRNA (5 nM). Densitometric furin/actin ratios are shown below the blots. (J) A549 cells were seeded in 60 mm dishes, and after 24 h, cells were transfected with the furin expression plasmid (4 μg) alone or co-transfected with RRM2 or control siRNA. The following day, the cells were infected with DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and subjected to western blot analysis under non-reducing conditions using anti-furin, anti-RRM2, anti-prM, anti-flavivirus E (4G2), and anti-actin antibodies. Densitometric prM/actin ratios are shown below the blots (left). Determination of viral maturation was carried out by calculating prM to E protein expression ratios normalized to those of control siRNA-transfected A549 cells. Detected signals of DENV E and prM proteins were quantified using ImageJ software as described in . prM-to-E ratios are plotted as bar graphs. Number indicates n -fold increase compared to Ctrl siRNA-transfected cells (right).

Article Snippet: Mouse anti-flavivirus envelope protein monoclonal antibody (D1-4G2-4-15) , ATCC , CVCL_J890.

Techniques: Knockdown, Quantitative RT-PCR, Infection, Standard Deviation, Western Blot, Expressing, Control, Software, Transfection, Glycoproteomics, SDS Page, Plasmid Preparation