Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Novel Chimeric Endolysin with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus

doi: 10.3389/fcimb.2017.00290

Figure Lengend Snippet: Bacterial species evaluated for sensitivity to CHAP-amidase.

Article Snippet: Escherichia coli , NDM 1 , Multi-drug resistant (ATCC BAA-2452) , Pakistan.

Techniques: Isolation

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37 proteins are receptors for FGF13A, related to Figure 3 (A) Representative plates of ELISA-based unbiased interactome screen next to measured values. The predicted extracellular sequence of LRRC37B (LRRC37B_ECTO) fused with alkaline phosphatase (ALP) was used as a bait; using LRRC37B_ECTO-ALP immobilized in each well of 384-well plates, 920 transmembrane or secreted proteins fused to a Fc domain were used as preys in each well: among them, only FGF13A-Fc was replicated and validated as a positive hit (blue wells vs. negative wells). (B) FGF13A co-immunoprecipitates the LRRC37B extracellular part (LRRC37B_ECTO) as well as its leucine-rich repeats (LRRs) but not the extracellular part devoid of the LRR from transfected HEK293T cells; the pictures and the left and right are from different gels from the same experiment. (C) IPs of human, chimpanzee, macaque LRRC37B proteins, or human LRRC37A2 protein from HEK293T cells transfected for their cDNA and FGF13A cDNA: all LRRC37 proteins except the macaque LRRC37B binds to FGF13A. (D) IPs of LRRC37B_ECTO and other transmembrane proteins (extracellular domain fused at the N-terminal with the prolactin leader peptide and an HA tag, and at the C-terminal with the transmembrane domain of PDGF-R) from HEK293T cells transfected for their cDNA and FGF13A cDNA; stars indicate the transmembrane protein in the input; the pictures and the left and right are from different gels from the same experiment. (E) Multi-angle light scattering with size-exclusion chromatography (SEC-MALS) of LRR recombinant protein showing a primary monomeric peak, with a low oligomeric fraction. (F) Binding assay of synthetic F13ExonS-biotin to transfected LRRC37B_ECTO (n = 6 experiments), transfected LRR_ECTO (n = 3), transfected ΔLRR_ECTO (n = 3), transfected display vector (n = 6), and non-transfected cells (n = 6) HEK293T cells (fitting curves for LRRC37B_ECTO and LRR_ECTO). (G) Binding assay of synthetic F13ExonS-biotin to transfected SCN8A (Nav1.6) (n = 11 experiments), transfected empty vector (n = 9), and non-transfected cells (n = 11) HEK293T cells (fitting curves for Nav1.6).

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Enzyme-linked Immunosorbent Assay, Sequencing, Transfection, Multi-Angle Light Scattering, Size-exclusion Chromatography, Recombinant, Binding Assay, Plasmid Preparation

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B is a receptor for FGF13A (A) FGF13 codes for several spliced isoforms; cell lysate and medium samples of HEK293T cells transfected for FGF13A, FGF13B, FGF13VY, and FGF13-core cDNAs (stars indicate each isoform in the cell extracts) detected with FGF13 antibody. (B) LRRC37B-HA IP from HEK293T cells co-transfected for LRRC37B-HA cDNA and cDNAs coding for the different FGF13 isoforms (stars indicate each isoform in the inputs) detected with FGF13 antibody. (C) LRRC37B-HA IP from HEK293T cells transfected for LRRC37B-HA cDNA and with recombinant FGF13A or its synthetic F13ExonS applied in the culture medium detected with a FGF13A-specific antibody (used in next panels). (D and E) IP from HEK293T cells transfected with LRRC37B and mutants lacking or carrying LRR of the extracellular domain and treated with recombinant FGF13A in the culture medium (stars indicate each LRRC37B protein in the inputs). (F) Fluorescence polarization (FP) assay between LRR recombinant protein and F13ExonS peptide, F13ExonU peptide (part of FGF13B) and two random peptides (n = 9 measures; mean ± SD). (G) IP of Nav1.6 (SCN8A) from HEK293T cells transfected for SCN8A cDNA with recombinant FGF13A or its synthetic F13ExonS applied in the culture medium. (H) Nav1.6 IPs from HEK293T cells transfected for SCN8A ± FGF13A ± LRRC37B cDNAs. (I) Schematics of the LRRC37B-FGF13A-Nav1.6 interaction. See also .

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Transfection, Recombinant, Fluorescence, FP Assay

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: FGF13A regulates neuronal excitability through its F13ExonS peptide (A) Examples traces of evoked AP of mouse CPNs (barrel cortex, P24–P32) with 50 nM recombinant FGF13A or synthetic F13ExonS extracellular application. (B) Corresponding firing rates (mean + SEM). (C) Dose-response effect of recombinant FGF13A extracellular application on mouse CPNs at 0 nM (11 neurons from 3 animals), 5 nM (8 neurons from 3 animals), 10 nM (9 neurons from 2 animals), and 50 nM (10 neurons from 4 animals) or synthetic F13ExonS at 50 nM (18 neurons from 8 animals) (values for each neuron are normalized to their initial value before application; lines at median; for each dose, paired Wilcoxon test). (D) Similar dose-response effects on rheobase. (E) Single evoked AP examples and dose-response effects on AP properties (shown as in C). (F) Phase plot analysis of AP generation (multiple trains, ) with 50 nM FGF13A or F13ExonS extracellular application on mouse CPNs (each replicate in blue or purple; mean + SEM in black; paired Wilcoxon tests). ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also .

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Recombinant

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: FGF13A act extracellularly, but not intracellularly, on neuronal excitability, related to Figure 4 (A and B) Electrophysiological properties complementary to Figure 4 , with treatment of mouse cortical sections (barrel cortex, layer 2/3 CPNs) with recombinant FGF13A extracellularly at 0 nM (11 neurons from 3 animals), 5 nM (8 neurons from 3 animals), 10 nM (9 neurons from 2 animals), and 50 nM (10 neurons from 4 animals) or synthetic F13ExonS at 50 nM (18 neurons from 7 animals) (values for each neuron are normalized to their initial value before application; lines at median; for each dose paired Wilcoxon test: all comparisons are ns). (C) IV-curves (ionic currents, mean + SEM) and normalized maximum currents (values normalized to values before application; lines at median; paired Wilcoxon tests for each dose) of mouse CPNs with extracellular application of recombinant FGF13A at 0, 5, 10, and 50 nM or synthetic F13ExonS extracellular application at 50 nM. (D) Phase plot analysis of AP generation in multiple AP trains from mouse neurons before and after FGF13A or F13ExonS 50 nM application complementary to Figure 4 F (see in ). (E) AP firing rate (left, mean + SEM; right, line at median; Mann-Whitney test) and rheobase (line at median; Mann-Whitney test) of mouse neurons (barrel cortex, layer 2/3 CPNs) with/without 50 nM intracellular application of recombinant FGF13A (control: 18 neurons from 7 animals; FGF13A: 8 neurons from 2 animals). (F) AP properties of mouse neurons with/without 50 nM intracellular application of recombinant FGF13A (lines at median; Mann-Whitney tests). (G) Electrophysiological properties of mouse neurons with/without 50 nM intracellular application of recombinant FGF13A (lines at median; Mann-Whitney tests). (H) Phase plot analysis of APs generation in multiple AP trains of mouse neurons with/without 50 nM intracellular application of recombinant FGF13A (lines at median; Mann-Whitney tests) (see in ). (I) IV-curves ionic currents) and maximum currents (lines at median; Mann-Whitney tests) of mouse neurons with/without 50 nM intracellular application of recombinant FGF13A (18 neurons from 7 animals for each condition). ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Recombinant, MANN-WHITNEY, Control

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B interacts with FGF13A following gain of function in the mouse cerebral cortex, related to Figure 5 (A) IP of LRRC37B-HA from mouse cortical protein extract (P17) of LRRC37B-HA/EGFP transfected mouse cortex compared with EGFP alone. (B) Quantification of FGF13A staining at the soma and AIS levels (27–28 neurons from 9 animals from 3 litters per condition; lines at median; Mann-Whitney tests) related to Figure 5 B. (C) Autocorrelation of the LRRC37B, FGF13A, and Navα signals (mean ± SEM) and corresponding periodicity (n = 21 neurons from 3 animals from 3 litters for the LRRC37B/FGF13A and for the LRRC37B/Navα stainings for control and LRRC37B neurons; line at mean) related to Figure 5 A. (D and E) Electrophysiological properties complementary to Figures 5 C–5G of mouse pyramidal neurons (layer 2/3 barrel cortex) transfected for LRRC37B/EGFP (15 neurons from 8 animals from 5 litters) or EGFP only (n = 13 neurons from 7 animals from 5 litters) before and after recombinant FGF13A protein 50 nM application (in gray and pink, each replicate; in dark and red, mean + SEM; paired Wilcoxon tests to test the application per group, Mann-Whitney tests to compare groups before and after applications). (F) Phase plot analysis of AP generation in multiple AP trains from control and LRRC37B-transfected mouse neurons before and after FGF13A 50-nM application complementary to Figure 5 G (see in ). (G) IV-curves (ionic currents; mean + SEM) and maximum currents of mouse pyramidal neurons transfected for LRRC37B/EGFP or EGFP only before and after with recombinant FGF13A protein 50 nM application (in gray and pink, each replicate; in dark and red, mean + SEM; paired Wilcoxon tests to test the application per group, Mann-Whitney tests to compare groups before and after applications). ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Transfection, Staining, MANN-WHITNEY, Control, Recombinant

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B concentrates FGF13A at the level of the AIS (A) AIS of LRRC37B and control CPNs immunostained for EGFP, LRRC37B, FGF13A, and Navα subunits (Nav channels). (B) LRRC37B and control CPNs immunostained for EGFP and FGF13A. (C) Examples of traces of evoked AP of control and LRRC37B neurons with 50-nM recombinant FGF13A extracellular application. (D and E) Corresponding firing rates (mean + SEM) of LRRC37B neurons (15 neurons from 8 animals from 5 litters) and control neurons (13 neurons from 7 animals from 5 litters) with corresponding quantification (D) and rheobase (E) (in gray and pink, each replicate; in dark and red, mean + SEM; paired Wilcoxon tests to test the application per group, Mann-Whitney tests to compare groups before and after applications). (F) Corresponding single evoked AP examples and properties (shown as in D and E). (G) Phase plot analysis of AP (multiple train) in LRRC37B neurons (11 neurons from 6 animals from 3 litters) and control neurons (10 neurons from 4 animals from 3 litters) with 50 nM FGF13A extracellular application (shown as in D and E) . ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01. See also .

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Control, Recombinant, MANN-WHITNEY

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B binds to SCN1B through its specific B domain (A and B) IP of different protein lacking or carrying the LB specific domain of the extracellular domain of LRRC37B from HEK293T cells co-transfected for the SCN1B cDNA. (C) SCN1B IPs from HEK293T cells transfected for SCN1B ± LRRC37B ± FGF13A cDNAs. (D) Nav1.6 IPs from HEK293T cells transfected for SCN8A ± SCN1B ± LRRC37B cDNAs. See also .

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Transfection

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet:

Article Snippet: FGF13A E. coli recombinant protein originates from Novus Biologicals (NBP2-35009).

Techniques: Recombinant, Plasmid Preparation, Magnetic Beads, Expressing, Comparison, Software

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: PNA probe hybridization assay for specific detection of uropathogens. A) We have designed PNA probes specific to the i) E. coli and ii) P. mirabilis species, the iii) Enterobacterales order, and the iv) eubacterial kingdom, in order to be able to detect all predominant uropathogens. We ensure that the designed probes can detect signal from target bacteria over blank urine background (N) by measuring probe fluorescence in the presence of E. coli (EC), P. mirabilis (PM), K. pneumoniae (KP), and P. aeruginosa (PA) ( p ‐values are calculated using unpaired one‐tailed t ‐tests; * p < 0.05, ** p < 0.01, *** p < 0.001, no asterisks between bars indicates no significant difference). B) Our assay works across a wide range of i) lysis temperatures and ii) hybridization temperatures (green: selected temperatures). C) Bulk‐based pheno‐molecular AST of reference E. coli ATCC 25922 and multi‐drug resistant E. coli BAA 2471 using hybridization detection of 16S rRNA is feasible, but requires >90 min of culture/antibiotic exposure to differentiate the effect of gentamicin on the susceptible and the resistant strains of E. coli . Data presented as mean +/− SD, n ≥ 3 or n ≥ 2 (bulk pheno‐molecular AST).

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Hybridization, Bacteria, Fluorescence, One-tailed Test, Lysis

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: Single‐cell detection of bacterial 16S rRNA from urine using microfluidic droplets. A) i) Urine samples of distinctly different color and turbidity can be discretized using flow‐focusing to generate monodisperse droplets (scale bars ≈50 µm) of ii) 4 ± 1 pL volume. B) Droplet fluorescence peak traces i) without E. coli , droplets emit baseline fluorescence signal, and have a positive droplet rate of 0.0079% (also known as the average limit of blank). ii) In the presence of 10 7 CFU mL −1 E. coli , droplets emit a higher fluorescence signal, and have a positive droplet rate of 6.67%. C) Droplet‐based quantification of E. coli in urine across four orders of magnitude within the clinically relevant dynamic range for UTIs (10 4 to 2 × 10 7 CFU mL −1 ), R 2 = 0.992 D) i) Reduction in droplet volume from 30 to 4 to 1 pL results in lower background fluorescence signals (scale bars in white ≈100 µm). ii) Compared to larger 30 pL droplets, 4 pL droplets facilitate faster generation of differentiable fluorescence signal over the reduced local background, as quickly as within 15 min. Data in (C,D(i)) presented as mean +/− SD, n ≥ 2 except for 2 × 10 7 CFU mL −1 input bacterial concentration in (C).

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Fluorescence, Concentration Assay

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: Accelerating antimicrobial susceptibility assessment via quantitative measurement of 16S rRNA from single cells. A) LIF detection of droplets containing E. coli cells suspended in MH broth i) without 30 min culture results in the expected 8% positive droplet frequency (7.02% observed), ii) following 30 min culture results in higher positive droplet intensities (indicative of higher 16S rRNA production) and 7.70% frequency, and iii) and after 30 min culture along with bactericidal gentamicin results in lower positive droplet intensities (indicative of relatively lower 16S rRNA production) and 3.12% frequency. B) Resistant E. coli can be differentiated from reference E. coli spiked into urine by comparing the positive droplet percentage from cells subject to antibiotic and no‐antibiotic conditions (“Normalized Positive Droplet Population”) for culture/drug exposure durations as low as 10 min. C) Resistant and susceptible strains of E. coli can be differentiated using our platform for three different antibiotics spanning distinct classes—gentamicin (aminoglycoside), ciprofloxacin (fluoroquinolone), and ampicillin (beta lactam). Error bars represent 1 standard deviation. The p ‐values are calculated from unpaired one‐tailed t ‐tests.

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Standard Deviation, One-tailed Test

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: DropDx clinical comparison study of 50 deidentified patient samples from Johns Hopkins Hospital. A) Each sample was simultaneously tested using clinical standard ID/AST tests as well as with 2 DropDx devices for measurements without and with ciprofloxacin. For ID, we used a combination of EC, EB, and UNI probes. B) Our 7‐outcome DropDx workflow is used to determine if there is a Gram‐negative bacterial infection present, whether the infecting pathogen is E. coli , whether the infecting pathogen is in the Enterobacterales order, or whether the infecting pathogen is a different (Gram‐negative) bacteria and to assess the susceptibility of the infecting pathogen to ciprofloxacin. λ is the proportion of droplets that contain a single cell to all droplets. C) Unbiased thresholding for each diagnostic metric was conducted in pilot studies using ROC curve analysis, and the final data groups and resulting ROC curves are plotted for i) differentiating culture‐positive from culture‐negative samples (AUC: 0.964), for ii) differentiating E. coli from non‐ E. coli samples (AUC: 1.000), for iii) differentiating Enterobacterales from non‐ Enterobacterales samples (AUC: 0.956), and for D) differentiating ciprofloxacin resistant from susceptible samples (AUC: 0.951). Importantly, DropDx's single‐cell pheno‐molecular AST results in a categorical agreement of 95.3% with no major errors. Error bars represent mean and standard error. The p ‐values are calculated from unpaired one‐tailed t‐tests.

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Comparison, Infection, Bacteria, Diagnostic Assay, One-tailed Test