Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: Cellular metabolism in human myotubes after miR-136-3p transfection or NRDC silencing. Mitochondrial respiration in miR-136-3p-transfected or NRDC- silenced human myotubes was monitored using the Mitochondrial Stress Test. (A) OCR and (B) ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyzer. The trace shows representative data. (C) Quantification of the mitochondrial respiration data for basal respiration, maximal respiration, ATP production, and spare respiratory capacity obtained from 3 independent experiments. Human myotubes were transfected with miR-136-3p or siRNA against NRDC before determination of (D) uptake of radiolabeled glucose, (E) rates of radiolabeled glucose oxidation, (F) conversion of radiolabeled glucose into glycogen, (G) rate of radiolabeled palmitic acid oxidation, (H) protein synthesis as assessed by incorporation of puromycin, and (I) lactate release into the media. Results are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.005 vs. control cells. ECAR = extracellular acidification rate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; ns = no significance; OCR = oxygen consumption rate; OigoA = oligomycin A; Rot/AA = rotenone and antimycin A; si NRDC = small interfering RNA of NRDC; siRNA = small interfering RNA; scr = negative control for small interfering RNA.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Transfection, Control, Negative Control, Small Interfering RNA

Journal: Antibody Therapeutics

Article Title: An IgG-like fusion protein comprising an anti-spike S2 antibody and ACE2 exhibits potent and broad neutralization against SARS-CoV-2 and variants of concern

doi: 10.1093/abt/tbag041

Figure Lengend Snippet: Characterization of the binding and neutralizing properties of P23 compared with reported anti-S2 mAbs. (A) Competitive inhibition of S2P6, Fp.006, and CV3-25 (hIgG1 format) and CC40.8, 76E1, and 54043-5 (scFv-hFc format) binding to the SARS-CoV-2 spike protein by P23 mIgG2a was assessed using competitive ELISA. NC, negative control (PBS for S2P6, Fp.006, and CV3-25; control IgG for CC40.8, 76E1, and 54043-5). OD 450 –OD 630 values are plotted against P23 concentration. (B) Cross-binding activity assessed by flow cytometry. Expi293F cells expressing full-length spike proteins from SARS-CoV-2 (WT, D614G, and JN.1), bat-CoV RaTG13, pangolin-CoV, MERS-CoV, HCoV-HKU1, or SARS-CoV-1 were stained with 10 μg/ml of each indicated antibody. (C) Neutralization activity of P23, S2P6, Fp.006, and CV3-25 against lentivirus-based pseudoviruses of WT SARS-CoV-2, SARS-CoV-2 D614G, and pangolin-CoV. (D) Neutralization activity of P23, S2P6, Fp.006, and CV3-25 against the VSV-based pseudoviruses of WT SARS-CoV-2. (E) IC 50 values corresponding to (C) and (D). n.m., not measurable.

Article Snippet: The corresponding coding sequences were cloned into expression vectors and co-transfected with a BirA ligase-expressing vector into Expi293F cells using polyethyleneimine (PEI, Polysciences, Cat. 23966).

Techniques: Binding Assay, Inhibition, Competitive ELISA, Negative Control, Control, Concentration Assay, Activity Assay, Flow Cytometry, Expressing, Staining, Neutralization

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: A) Binding of IgG and Fab to recombinant RBDs of Sarbecoviruses from different clades by ELISA including SARS-CoV-2 (clade 1b), WIV-1 (clade 1a), Rs4321 (clade 1a), RacCS203 (clade 2), Rc-o319 (clade 5), and RhGB07 (clade 3). Experiments were run in duplicate and repeated twice. A representative assay is shown. Fab binding is shown with the dotted line and IgG binding with the full line. B) Neutralising activity of IgGs against SARS-CoV-2 and variants of concern as well as SARS-CoV-1 and WIV-1. C) IC 50 values for neutralisation against SARS-CoV-2 variants of concern and other sarbecoviruses that utilise human-ACE2. For A–C, the mean value is plotted with error bars showing ± standard deviation (s.d.). D) Ability of IgGs to inhibit the interaction between cell surface human ACE2 and soluble SARS-CoV-2 and WIV-1 RBDs. IgGs were pre-incubated with fluorescently labelled RBD before addition to HeLa-ACE2 cells. The percentage reduction in mean fluorescence intensity is reported. Experiments were performed in duplicate. E) Superimposed 3D structures of the V1WT_06 (dark blue), V1WT_41 (purple), and VA14_26 (red) around WIV-1 RBD (light blue, from PDB 8WLZ). The data underlying this Figure can be found in .

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Incubation, Fluorescence

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: A) A closeup view of the superimposed structure of V1WT_06 bound to WIV-1 RBD on a trimeric spike (from PDB: 8WLZ). A clash with NTD of chain B is observed when V1WT_06 Fab-RBD complex is aligned onto RBD in the up conformation (chain A) in trimeric WIV-1 spike protein. Notably, the binding site is inaccessible in the RBD-down conformation. B) V1WT_06 heavy chain only interaction with WIV-1 RBD (grey). Heavy and light chains are shown with the dark and light blue ribbons, respectively. C) V1WT_06 epitope footprint overlayed with epitope footprints of other site V neutralising antibodies including S2H97, CC25.4, 5817, R1-32, and E465 patch [ , – , ]. SARS-CoV-2 RBD (from PDB: 6m0j) is coloured based on the amino acid conservation across 87 bat sarbecoviruses [ , , , ]. D) Sequence alignment of a panel of bat sarbecovirus RBDs. Contact residues of V1WT_06 mAb on the WIV-1-RBD are indicated with black dots. The colouring scheme describes the percent identity (PID) between the sequences as implemented in Jalview 2.11.4.0. E) Comparison in neutralisation potency of V1WT_06 Fab and IgG against SARS-CoV-2 (D614G), SARS-CoV-1, and WIV-1. Mean values are plotted with error bars showing ± standard deviation (s.d.). The data underlying this Figure can be found in .

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Binding Assay, Sequencing, Comparison, Standard Deviation

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: A) VA14_26 and V1WT_41 antibody footprints overlayed with the epitope footprints of other RBD class 4 neutralising antibodies including CR3022, 10−40, ADI-62113 [ – ]. SARS-CoV-2 RBD (from PDB: 6m0j) is coloured based on the amino acid conservation across 87 bat sarbecoviruses [ , , , ]. B) Superimposed 3D structure depicting the binding of VA14_26 (Red) to WIV-1 RBD (blue) in relation to ACE2 (orange, from PDB: 8WLZ) and CR3022 (pink, from PDB: 6W41). C) Superimposed 3D structure depicting the binding of V1WT_41 (green) to WIV-1 RBD (blue) in relation to ACE2 (orange, from PDB: 8WLZ). D) Sequence alignment of a panel of bat sarbecovirus RBDs. Contact residues for VA14_26 and V1WT_41 are indicated by red and pink dots and outline, respectively. Shared contact residues are shown with yellow dots and outline. E) Sequence alignment of a panel of SARS-CoV-2 variant RBDs. Contact residues for VA14_26 and V1WT_41 are indicated by red and pink dots and outline, respectively. Shared contact residues are shown with yellow dots and outline. The colouring scheme describes the percent identity (PID) between the sequences as implemented in Jalview 2.11.4.0. F) A close-up view of YYDRSG motif in V1WT_41 HCDR3 (green) in contact with WIV-1 RBD (grey), shown in stick representation, renumbered based on alignment to SARS-CoV-2 [ , ]. Presence of G109 breaks the H-bonding pattern in the beta hairpin to create a G1 β-bulge. This further enables D106 side chain to make hydrogen bond with the amides in the protein backbone, thereby stabilising the local structure. Combined this accentuates a right-handed twist found in G1 β-bulge . Twisting of the β-hairpin is thought to contribute towards stabilisation of RBD-Fab interaction in several other public mAbs with YYDRxG motif [ , ].

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Binding Assay, Sequencing, Variant Assay

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: Neutralisation of sarbecoviruses through entry via different host ACE2s. Neutralisation of A) Rc-o319 and B) RhGB07 was measured against entry via Rh. cornutus ACE2 and Rh. alcyone ACE2, respectively, which were transiently expressed in HEK293T/17 cells. C) Neutralisation of WIV-1 was compared between entry via hu-ACE2 (dotted line and closed symbols) and Rh. alcyone ACE2 (full line and closed symbols). Neutralisation was measured using HEK 293T cells transiently expressing either Human ACE2 or Rh. alcyone ACE2. For A-C, mean values are plotted with error bars showing ± standard deviation (s.d.). D) Ability of IgGs to inhibit the interaction between cell surface ACE2 (including hu-ACE2, Rh. cornutus ACE2 and Rh. alcyone ACE2) and soluble RBD (including SARS-CoV-2, WIV-1, Rs4231, RhBG07, and Rc-o319 RBDs). IgGs were pre-incubated with fluorescently labelled RBD before addition to ACE2 expressing cells. N.B = No binding of IgG to the RBD. The percentage reduction in mean fluorescence intensity is reported. Experiments were performed in duplicate. E) Antibody dependant cellular cytotoxicity (ADCC) of IgGs measured through the surface expression of CD107a on activated NK cells (NK-92 cells expressing CD16) . HIV-1 specific neutralising antibody, PGT128 is used as a negative control . The data underlying this Figure can be found in .

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Expressing, Standard Deviation, Incubation, Binding Assay, Fluorescence, Negative Control