Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss.

doi: 10.1073/pnas.0508053102

Figure Lengend Snippet: Fig. 1. Exposure to gentamicin in utero induces, and LCAR prevents, hearing loss and cochlear damage in newborn guinea pigs. (a) LCAR significantly decreases gentamicin-induced neonatal mortality (gentamicin vs. LCAR plus gentamicin, P 0.003). (b) Typical ABR traces in response to 10-ms clicks. (c) ABR experiments indicate that LCAR prevents the significant shift in hearing threshold associated with exposure to gentamicin. (d) Percentage of lost OHCs in the first, second, and third cochlear turns in newborn guinea pigs. Fourth turn data were not included because of the different nature of the damage (see Results and Discussion and f). (e) SEM image of a full guinea pig cochlea with the smaller, apical fourth turn on the left, and the bigger, basal first turn to the right. (f) Typical appearance of the OHCs in the apical fourth turn of animals exposed to gentamicin. The pattern of distribution is disrupted, many cells are lost, and others show atypical hair bundles. A single giant stereocilia is also visible (arrowhead). (g–i) SEM and confocal images of more basal turns show the normal structure of the organ of Corti, with three rows of OHCs and scars replacing dead cells (arrows).

Article Snippet: Gentamicin uptake was monitored with a monoclonal antibody against gentamicin (Fitzgerald Industries International, Chelmsford, MA).

Techniques: In Utero

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss.

doi: 10.1073/pnas.0508053102

Figure Lengend Snippet: Fig. 2. HEI-OC1 cells are sensitive to gentamicin. (a) HEI-OC1 cells quickly incorporate gentamicin, reaching a plateau at 6 h incubation. Gentamicin uptake is not prevented by preincubation with LCAR. (b and c) Gentamicin- filled vesicles (green) accumulate preferentially in the perinuclear region (b), and later distribute in the entire cytoplasm (c). (d and e) Labeling with anti-annexin V antibodies (green) and propidium iodide stain (red) indicates that many gentamicin-exposed cells undergo apoptosis (e), but necrotic (an- nexin V-negative, propidium iodide-positive) cells were also observed (d). (f and g) SEM studies indicate that plasma membrane blebbing (g) was signifi- cantly more frequent in cells exposed to gentamicin than in control cells (f).

Article Snippet: Gentamicin uptake was monitored with a monoclonal antibody against gentamicin (Fitzgerald Industries International, Chelmsford, MA).

Techniques: Incubation, Labeling, Staining, Clinical Proteomics, Membrane, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss.

doi: 10.1073/pnas.0508053102

Figure Lengend Snippet: Fig. 3. Gentamicin induces, and LCAR prevents, transcriptional up-regulation of Hrk. (a and b) Microarray results were validated by RT-PCR (a) and Western blot (b). (c) Confocal images of HEI-OC1 cells triple-labeled with anti-gentamicin (green), anti-Hrk (red), and the nuclear stain DAPI (blue) show more abundant Hrk expression in cells that incorporate than in those that do not incorporate gentamicin (arrowhead). (d) siRNA experiments confirm that Hrk expression is necessary for gentamicin-induced apoptosis. All of the siRNA oligonucleotides inhibit the expression of Hrk and its up-regulation by gentamicin, and prevent caspase-3 activation. These effects were abolished by cotransfection with Hrk-resistant (rHrk) cDNA.

Article Snippet: Gentamicin uptake was monitored with a monoclonal antibody against gentamicin (Fitzgerald Industries International, Chelmsford, MA).

Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Labeling, Staining, Expressing, Activation Assay, Cotransfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss.

doi: 10.1073/pnas.0508053102

Figure Lengend Snippet: Fig. 4. Gentamicin-induced up-regulation of Hrk is mediated by MAPKs. (a) Caspase-3 activation experiments indicate that gentamicin-induced apoptosis of HEI-OC1 cells is prevented by preincubation with LCAR and ERK12 inhibition, but enhanced by inhibition of JNK. JNK inhibition also interferes with the preventive effect of LCAR. Values are normalized (control 100%). (b) RT-PCR results confirmed that the effects of the inhibitors of MAPK on caspase-3 activation were associated with changes in Hrk expression. (c) Phosphorylation studies indicate that gentamicin activates ERK12 and inactivates JNK. LCAR, in turn, is able to reverse the gentamicin-induced inactivation of JNK. (d–f) Confocal microscopy of HEI-OC1 cells triple-labeled with gentamicin (green), anti-p-ERK (red), and DAPI (blue) confirms that gentamicin activates ERK and demonstrates that gentamicin also induces its translocation to the nucleus. A cell with numerous gentamicin-filled vesicles in the cytoplasm and p-ERK concentrated in the nucleus is pointed out with an arrow. An arrowhead indicates a cell that incorporates only a small amount of gentamicin and shows cytoplasmic labeling with anti-p-ERK.

Article Snippet: Gentamicin uptake was monitored with a monoclonal antibody against gentamicin (Fitzgerald Industries International, Chelmsford, MA).

Techniques: Activation Assay, Inhibition, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Phospho-proteomics, Confocal Microscopy, Labeling, Translocation Assay

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: Orthopedic implant-related infection animal model of cocktail of isobavachalcone and curcumin with gentamycin against MRSA. (A) In vivo study design. (B) Surgical procedures of distal femoral implant-related infection mouse model. 1–5 refer to: (1) Distal femoral implant using a trans-knee approach, after incise internally alongside patella and externally dislocating patella to expose the distal femoral articular surface, and the entry point, intercondylar notch, was shown by the yellow arrow. (2) 25-gauge needle was used to open a corridor into the femoral medullary canal. (3) The customized 0.6 mm diameter needle was implanted after the inoculation of 5 μl MRSA USA300 inoculum suspension. (4) The caudal end of the implant was left about 1 mm outside the femur after implantation. (5) The representative X-ray image of implant well positioned in the medullary canal and distal femur. (C) Body weight of animals throughout in vivo experiment. (D) Representative appearance of distal femurs of mice 28 days after intraperitoneal administration of different groups of antimicrobials combinations, gentamicin served as control. I.P., intraperitoneal injection; GEN, Gentamycin; ISB, Isobavachalcone; CRM, Curcumin.

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: Infection, Animal Model, In Vivo, Suspension, Control, Injection

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: Interaction of Isobavachalcone and Curcumin with Antibiotics on MSSA and MRSA strains. (A,B) Heatmaps of fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI) of combination of gentamycin with isobavachalcone or curcumin, darker orange color indicates interaction is superimposed or synergistic. (C,D) FICI and FBCI of Isobavachalcone and Curcumin with antibiotics against Staphylococcus aureus JAR strain, respectively. FICI ≤ 0.5 represents synergy, FICI > 0.5–4 represents no synergistic interaction, and FICI > 4.0 represents antagonism. FICI: fractional inhibition concentration index; FBCI: fractional bactericidal concentration index; DAP: Daptomycin with 50 μg/ml Ca2+; RIF: Rifampicin; GEN: Gentamycin; LEV: Levofloxacin; ISB: Isobavachalcone; CRM: Curcumin; MSSA: Methicillin-susceptible Staphylococcus aureus ; MRSA: Methicillin-resistant Staphylococcus aureus .

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: Interaction of isobavachalcone and curcumin with Gentamycin against Staphylococcus aureus JAR biofilm. (A) Residual S. aureus JAR on Biofilm challenged by combination of Gentamycin with Isobavachalcone or Curcumin. (B) Coefficient of Drug Interaction (CDI) of the combination of Gentamycin with isobavachalcone or curcumin. (C) Synergistic Combination of isobavachalcone with Gentamycin, and Curcumin with Gentamycin. CDI < 1, =1 or >1 indicate synergy, superposition or antagonism, respectively, CDI < 0.7 indicates significant synergism. Data were presented as mean ± SD. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.001, when compared to ISB/CRM with the same Gentamycin concentration, ns represents no significance. GEN, Gentamycin; ISB, Isobavachalcone; CRM, Curcumin; CDI, Coefficient of Drug Interaction.

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: EUCAST checkerboard assay showed isobavachalcone and curcumin interact synergistically with presence of gentamycin against planktonic Staphylococcus aureus . (A,B) Heatmaps of fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI) of combination of isobavachalcone and curcumin, darker orange color indicates interaction is superimposed or synergistic. (C) FICI of isobavachalcone with curcumin against S. aureus JAR and USA300 strain. (D) FBCI of isobavachalcone with curcumin against S. aureus JAR and USA300 strain. FICI ≤ 0.5 represents synergy, FICI > 0.5–4 represents no synergistic interaction, and FICI > 4.0 represents antagonism. CDI < 1, =1 or >1 indicate synergy, superposition or antagonism, respectively, CDI < 0.7 indicates significant synergism. Data were presented as mean ± SD. FICI, fractional inhibition concentration index; FBCI, fractional bactericidal concentration index; GEN, Gentamycin; ISB, Isobavachalcone; CRM, Curcumin; CDI, Coefficient of Drug Interaction; MSSA, Methicillin-susceptible Staphylococcus aureus ; MRSA, Methicillin-resistant Staphylococcus aureus .

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: In vitro Eradication of Staphylococcus aureus biofilms by the GEN-ISB-CRM combination. (A,C) Quantification of residual viable cells on S. aureus JAR biofilm and USA300 biofilm, respectively. (B,D) Coefficient of Drug Interaction (CDI) shows synergy between Isobavachalcone and Curcumin with 128 μg/ml Gentamycin against S. aureus JAR biofilm and USA300 biofilm, respectively. (E) Representative characteristics of MRSA USA300 biofilm after 24 h challenge. SEM: biofilms cultured at 37°C for 24 h were treated with 128 μg/ml GEN (control), 128 μg/ml GEN + 125 μg/ml CRM, 128 μg/ml GEN + 6.25 μg/ml ISB, 128 μg/ml GEN + 125 μg/ml CRM + 6.25 μg/ml ISB for 24 h, respectively. All SEM images were captured at EHT = 10 kV, Mag = 5KX. CLSM: representative confocal laser scanning microscopy (100× oil immersion) images of LIVE/DEAD ® BacLight staining showing the synergistic effect of different drug combinations on USA300 biofilm challenged for 24 h, green: live cells; red: dead cells. Data were presented as mean ± SD. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.01 when compared to 0 μg/ml CRM with the same ISB concentration group, ns represents no significance. GEN, Gentamycin; ISB, Isobavachalcone; CRM, Curcumin; CDI, Coefficient of Drug Interaction; SEM, Scanning Electron Microscopy; CLSM, Confocal Laser Scanning Microscopy.

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: In Vitro, Cell Culture, Control, Confocal Laser Scanning Microscopy, Staining, Concentration Assay, Electron Microscopy

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: Micro Computed tomography (μCT) imaging suggests protection of cocktail therapy against osteolysis in orthopedic implant-related infections due to MRSA. (A) Representative X-ray images of distal femur of mouse with intramedullary implant. (B) Representative images of trans axial 3D reconstructed micro-architecture of distal femur around implant. (C) Bone morphometry in distal femur shows protection of combination of Isobavachalcone and Curcumin against osteolysis during orthopedic implant-related infection, related parameters including percent bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp), bone mineral density (BMD) and bone surface density (BS/TV) were measured, data were presented as mean ± SD, * represents p < 0.05 and ** represents p < 0.01 when compared to Gentamicin group; # represents p < 0.05 and ## represents p < 0.01 when GEN + ISB + CRM group compared to GEN + ISB/CRM group; GEN, 20 mg/kg/day Gentamycin; ISB, 20 mg/kg/day Isobavachalcone; CRM, 20 mg/kg/day Curcumin.

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: Micro-CT, Imaging, Infection

Journal: Frontiers in Microbiology

Article Title: Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

doi: 10.3389/fmicb.2022.958132

Figure Lengend Snippet: Cocktail therapy of Gentamicin with Isobavachalcone and Curcumin alleviate local tissue inflammation, enhance reduction of MDSC M1 Polarization and Eradication of MRSA biofilm in vivo . (A) HE and immunofluorescent staining of distal femur suggest reduction of tissue inflammation and M1 polarization of MDSC, green represents CD11b, a key marker to identify MDSC, while red represents CXCL10, a key marker of activated M1, he black dashed box indicates the approximate location of the implant, and the yellow dashed box range is the area where M1-polarized MDSC in the adjacent implant tissue is observed. (B) Quantification of remaining MRSA in bone with implant. (C) Flow cytometry analysis showed reduction of MDSC in peripheral blood by Gentamicin with ISB or CRM and cocktail therapy. (D) Gating strategy for flow cytometry analysis and representative analysis of frequency of MDSC in peripheral blood. All data were presented as mean ± SD, * represents p < 0.05 and ** represents p < 0.01 when compared to Gentamicin group; # represents p < 0.05 and ## represents p < 0.01 when GEN + ISB + CRM group compared to GEN + ISB/CRM group; GEN, 20 mg/kg/day Gentamycin; ISB, 20 mg/kg/day Isobavachalcone; CRM, 20 mg/kg/day Curcumin.

Article Snippet: Gentamicin sulfate (HY-A0276), Daptomycin (HY-B0108), Rifampicin (HY-B0272), and Levofloxacin (HY-B0330) were purchased from MedChemExpress, China.

Techniques: In Vivo, Staining, Marker, Flow Cytometry

Journal: STAR Protocols

Article Title: Breast cancer PDxO cultures for drug discovery and functional precision oncology

doi: 10.1016/j.xpro.2023.102402

Figure Lengend Snippet:

Article Snippet: Gentamicin , Genesee Scientific , cat# 25-533.

Techniques: Recombinant, Blocking Assay, Isolation, Viability Assay, Software, Cell Counting, Gentle, Adhesive, Microscopy, Transferring

Journal: STAR Protocols

Article Title: Breast cancer PDxO cultures for drug discovery and functional precision oncology

doi: 10.1016/j.xpro.2023.102402

Figure Lengend Snippet: PDxO base media

Article Snippet: Gentamicin , Genesee Scientific , cat# 25-533.

Techniques: Concentration Assay