Structured Review

Thermo Fisher acarbose
Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, <t>acarbose</t> and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively
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Images

1) Product Images from "Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention"

Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

Journal: NPJ Biofilms and Microbiomes

doi: 10.1038/s41522-018-0075-0

Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively
Figure Legend Snippet: Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

Techniques Used: Concentration Assay, Activity Assay

Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown
Figure Legend Snippet: Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

Techniques Used: Sequencing, Binding Assay, SPR Assay

Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability
Figure Legend Snippet: Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

Techniques Used: Mass Spectrometry, Incubation, Cell Culture, Alamar Blue Assay

Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p
Figure Legend Snippet: Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

Techniques Used: Crystal Violet Assay, Mass Spectrometry, Cell Culture, Incubation

2) Product Images from "Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model"

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model

Journal: PeerJ

doi: 10.7717/peerj.4788

Inhibitory effect of the methanolic extract of carob against α-glucosidase. The percentage of inhibition of the methanolic carob extract and α-acarbose against α-glucosidase. The IC 50 of the methanolic carob extract (97.13 ± 4.11 µg mL −1 ) against α-glucosidase was higher than that of α-acarbose (27.05 ± 0.99 µg mL −1 ).
Figure Legend Snippet: Inhibitory effect of the methanolic extract of carob against α-glucosidase. The percentage of inhibition of the methanolic carob extract and α-acarbose against α-glucosidase. The IC 50 of the methanolic carob extract (97.13 ± 4.11 µg mL −1 ) against α-glucosidase was higher than that of α-acarbose (27.05 ± 0.99 µg mL −1 ).

Techniques Used: Inhibition

Inhibitory effect of the methanolic extract of carob against α-amylase. The percentage of inhibition of the methanolic carob extract and α-acarbose against against α-amylase. The IC 50 of carob methanolic extract (92.99 ± 0.22 µg mL −1 ) was higher than that of α-acarbose (23.33 ± 0.73 µg mL −1 ) against α-amylase.
Figure Legend Snippet: Inhibitory effect of the methanolic extract of carob against α-amylase. The percentage of inhibition of the methanolic carob extract and α-acarbose against against α-amylase. The IC 50 of carob methanolic extract (92.99 ± 0.22 µg mL −1 ) was higher than that of α-acarbose (23.33 ± 0.73 µg mL −1 ) against α-amylase.

Techniques Used: Inhibition

3) Product Images from "Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention"

Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

Journal: NPJ Biofilms and Microbiomes

doi: 10.1038/s41522-018-0075-0

Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively
Figure Legend Snippet: Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

Techniques Used: Concentration Assay, Activity Assay

Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb PpiB (Rv2582) exhibits homology with proteins from other biofilm forming bacteria and possesses similar amino acids Arg and Pro at the binding site of cyclosporine-A (highlighted in green box) and acarbose (highlighted in red box), respectively. A dimer of atomic gallium 17 similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown
Figure Legend Snippet: Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb PpiB (Rv2582) exhibits homology with proteins from other biofilm forming bacteria and possesses similar amino acids Arg and Pro at the binding site of cyclosporine-A (highlighted in green box) and acarbose (highlighted in red box), respectively. A dimer of atomic gallium 17 similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

Techniques Used: Sequencing, Binding Assay, SPR Assay

Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability
Figure Legend Snippet: Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

Techniques Used: Mass Spectrometry, Incubation, Cell Culture, Alamar Blue Assay

Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p
Figure Legend Snippet: Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

Techniques Used: Crystal Violet Assay, Mass Spectrometry, Cell Culture, Incubation

4) Product Images from "Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model"

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model

Journal: PeerJ

doi: 10.7717/peerj.4788

Inhibitory effect of the methanolic extract of carob against α-amylase. The percentage of inhibition of the methanolic carob extract and α-acarbose against against α-amylase. The IC 50 of carob methanolic extract (92.99 ± 0.22 µg mL −1 ) was higher than that of α-acarbose (23.33 ± 0.73 µg mL −1 ) against α-amylase.
Figure Legend Snippet: Inhibitory effect of the methanolic extract of carob against α-amylase. The percentage of inhibition of the methanolic carob extract and α-acarbose against against α-amylase. The IC 50 of carob methanolic extract (92.99 ± 0.22 µg mL −1 ) was higher than that of α-acarbose (23.33 ± 0.73 µg mL −1 ) against α-amylase.

Techniques Used: Inhibition

5) Product Images from "Reconstruction and in silico analysis of an Actinoplanes sp. SE50/110 genome-scale metabolic model for acarbose production"

Article Title: Reconstruction and in silico analysis of an Actinoplanes sp. SE50/110 genome-scale metabolic model for acarbose production

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00632

Biosynthetic pathways of acarbose in Actinoplanes sp. SE50/110 . The genes mal EFG in the pathway were ACPL_6402, ACPL_3200, ACPL_4618, ACPL_5585, ACPL_6401, ACPL_6726, ACPL_3782, ACPL_5386, and ACPL_6400.
Figure Legend Snippet: Biosynthetic pathways of acarbose in Actinoplanes sp. SE50/110 . The genes mal EFG in the pathway were ACPL_6402, ACPL_3200, ACPL_4618, ACPL_5585, ACPL_6401, ACPL_6726, ACPL_3782, ACPL_5386, and ACPL_6400.

Techniques Used:

Effect of amino acids on cell growth rate and acarbose production rate .
Figure Legend Snippet: Effect of amino acids on cell growth rate and acarbose production rate .

Techniques Used:

Effects of different aeration rates on acarbose production .
Figure Legend Snippet: Effects of different aeration rates on acarbose production .

Techniques Used:

Percentage of essential genes in each subsystem under different cultivated conditions. (A) Acarbose-synthesis medium. (B) Sucrose medium. The abbreviations of each subsystem are the same with that in Figure 2 .
Figure Legend Snippet: Percentage of essential genes in each subsystem under different cultivated conditions. (A) Acarbose-synthesis medium. (B) Sucrose medium. The abbreviations of each subsystem are the same with that in Figure 2 .

Techniques Used:

Chemical structure for acarbose .
Figure Legend Snippet: Chemical structure for acarbose .

Techniques Used:

Related Articles

High Performance Liquid Chromatography:

Article Title: Optimization of Phlorotannins Extraction from Fucus vesiculosus and Evaluation of Their Potential to Prevent Metabolic Disorders
Article Snippet: HPLC grade acetone, ethanol, methanol, n -hexane, ethyl acetate, acetonitrile, dimethylsulfoxide, hydrochloric acid, glacial acetic acid, sodium chloride, sodium hydroxide, potassium hydroxide, sodium and potassium tartarate, tris-HCl, and starch were acquired from Fisher (Pittsburgh, PA, USA). .. Dinitrosalycilic acid and Acarbose were purchased from Acros Organics (Hampton, NH, USA), calcium chloride from ChemLab (Eernegem, Belgium) and orlistat from AlfaAesar (Ward Hill, MA, USA).

Article Title: Reconstruction and in silico analysis of an Actinoplanes sp. SE50/110 genome-scale metabolic model for acarbose production
Article Snippet: .. The acarbose concentration was measured by HPLC using a Thermo Fisher Scientific (Waltham, MA, USA) Hypersil APS-2 NH2 column (250 × 4.6 mm, 5 μm). ..

Filtration:

Article Title: Reconstruction and in silico analysis of an Actinoplanes sp. SE50/110 genome-scale metabolic model for acarbose production
Article Snippet: Analytical methods The dry cell weight (DCW) was measured by filtration: 15 mL of culture was filtered via a vacuum pump, followed by washing three times with distilled water and drying at 70°C overnight to a constant weight. .. The acarbose concentration was measured by HPLC using a Thermo Fisher Scientific (Waltham, MA, USA) Hypersil APS-2 NH2 column (250 × 4.6 mm, 5 μm).

In Vitro:

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model
Article Snippet: Paragraph title: In vitro inhibitory effect of carob against α -amylase and α -glucosidase ... Acarbose 95% (Acros Organic, USA) dissolved in 30% DMSO was used as a positive control.

Positive Control:

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model
Article Snippet: .. Acarbose 95% (Acros Organic, USA) dissolved in 30% DMSO was used as a positive control. .. The reference sample contained the same reaction mixture except the same amount of test sample solution and positive control that were replaced by phosphate buffer solution.

Northern Blot:

Article Title: Optimization of Phlorotannins Extraction from Fucus vesiculosus and Evaluation of Their Potential to Prevent Metabolic Disorders
Article Snippet: Materials Grounded F. vesiculosus samples from July 2017 were purchased from Algaplus Lda (production site located at Ria de Aveiro coastal lagoon, Northern Portugal, 40°36′43″ N, 8°40′43″ W) an enterprise dedicated to the production of edible seaweeds in an integrated multi-trophic aquaculture (IMTA) system. .. Dinitrosalycilic acid and Acarbose were purchased from Acros Organics (Hampton, NH, USA), calcium chloride from ChemLab (Eernegem, Belgium) and orlistat from AlfaAesar (Ward Hill, MA, USA).

Incubation:

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model
Article Snippet: St Louis, USA) and 50 µL of each test sample solution (1 mg mL−1 extract) dissolved in 30% DMSO were added in each well and incubated at 25 °C for 10 min. Then, 50 µL of 4-Nitrophenyl (3-D-glucopyranoside (P -NPG) substrate solution (5 mM of P -NPG (Sigma-Aldrich, St Louis, USA) 7.53 mg/5mL dissolved in pH 6.9 phosphate buffer which prepared by mixing with 100 mM monobasic potassium phosphate); was added to the reacting mixture and re-incubated for 5 min at 25°C. .. Acarbose 95% (Acros Organic, USA) dissolved in 30% DMSO was used as a positive control.

Spectrophotometry:

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model
Article Snippet: Acarbose 95% (Acros Organic, USA) dissolved in 30% DMSO was used as a positive control. .. The absorbance of the reaction was recorded on UV-Visible spectrophotometry (Infinite M 200; Tecan, Switzerland) at 405 nm.

Concentration Assay:

Article Title: Reconstruction and in silico analysis of an Actinoplanes sp. SE50/110 genome-scale metabolic model for acarbose production
Article Snippet: .. The acarbose concentration was measured by HPLC using a Thermo Fisher Scientific (Waltham, MA, USA) Hypersil APS-2 NH2 column (250 × 4.6 mm, 5 μm). ..

Crystal Violet Assay:

Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention
Article Snippet: Paragraph title: Crystal violet assay ... Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

Activity Assay:

Article Title: Evaluation of the glycemic effect of Ceratonia siliqua pods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model
Article Snippet: Regarding α-glucosidase inhibitory activity of carob methanolic extract, it was assessed spectrophotometrically according to with few modifications. .. Acarbose 95% (Acros Organic, USA) dissolved in 30% DMSO was used as a positive control.

Cell Culture:

Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention
Article Snippet: .. Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India). .. At the end of 7 days of static phase culture, the media beneath the pellicle were aspirated out and remaining solid pellicle was stained by adding 125 µl (w/v) 0.1% Crystal Violet solution.

Mass Spectrometry:

Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention
Article Snippet: .. Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India). .. At the end of 7 days of static phase culture, the media beneath the pellicle were aspirated out and remaining solid pellicle was stained by adding 125 µl (w/v) 0.1% Crystal Violet solution.

Staining:

Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention
Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India). .. At the end of 7 days of static phase culture, the media beneath the pellicle were aspirated out and remaining solid pellicle was stained by adding 125 µl (w/v) 0.1% Crystal Violet solution.

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  • 99
    Thermo Fisher anti glucose transporter 1
    Elevated GLUT1 is associated with low Nuc-pYStat5 in human breast cancer and xenografts. (A)  Representative images from a breast cancer progression tissue array co-stained with anti-cytokeratins, DAPI, anti-pYStat5 or anti-GLUT1.  (B)  Expression of GLUT1 and nuclear pY-Stat5 in each tissue samples were quantified by AQUA and plotted. The range of normal GLUT1 expression is labeled on the plot by mean of normal mammary gland GLUT1 (log) score ± 3 SD.  (C)  Comparison of AQUA scores Nuc-pYStat5, Stat5a, Stat5b and PrlR between GLUT1 negative and positive breast cancer samples ( n  = 88) by Student’s  t  test.  (D)  IHC staining of LDH5, GLUT1, PrlR and Stat5 with consecutive slides of a representative T47D xenograft tumor.  (E)  Co-staining of pYStat5 and GLUT1 in representative T47D xenografts from mice that were injected intraperitoneally with vehicle or human prolactin. Red, pYStat5; green, GLUT1. AQUA, automated quantitative analysis; GLUT1, glucose transporter 1; IHC, immunohistchemistry; LDH5, lactate dehydrogenase-5; Nuc-pYStat5, nuclear localized and tyrosine phosphorylated Stat5; PrlR, prolactin receptor; SD, standard deviation.
    Anti Glucose Transporter 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti glucose transporter 1/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti glucose transporter 1 - by Bioz Stars, 2020-03
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    79
    Thermo Fisher gutnick glucose
    Effect of inhibiting the RNA futile cycle on the antibiotic tolerances of MazF persisters. E. coli MO:: mazE - mazF was diluted in <t>Gutnick-glucose</t> medium supplemented with aTc, cultured for 3 h, and treated with no inhibitor or Rif. One hour posttreatment, cells were exposed to no antibiotics (A), Amp (B), Ofl (C), Kan (D), or Strep (E). Rif treatment caused reduced culturability at time zero, which was regained during the course of the assay (refer to Fig. S6 in the supplemental material for supporting data). In MazF-accumulating cells, Rif treatment did not alter Amp or Ofl persister levels compared with the untreated control. However, MazF-accumulating cells were sensitive to Kan and Strep, and an increase in persister levels was observed only when transcription and, as a consequence, translation were inhibited with Rif treatment.
    Gutnick Glucose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gutnick glucose/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gutnick glucose - by Bioz Stars, 2020-03
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    96
    Thermo Fisher acarbose
    Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, <t>acarbose</t> and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively
    Acarbose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/acarbose/product/Thermo Fisher
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    acarbose - by Bioz Stars, 2020-03
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    Image Search Results


    Elevated GLUT1 is associated with low Nuc-pYStat5 in human breast cancer and xenografts. (A)  Representative images from a breast cancer progression tissue array co-stained with anti-cytokeratins, DAPI, anti-pYStat5 or anti-GLUT1.  (B)  Expression of GLUT1 and nuclear pY-Stat5 in each tissue samples were quantified by AQUA and plotted. The range of normal GLUT1 expression is labeled on the plot by mean of normal mammary gland GLUT1 (log) score ± 3 SD.  (C)  Comparison of AQUA scores Nuc-pYStat5, Stat5a, Stat5b and PrlR between GLUT1 negative and positive breast cancer samples ( n  = 88) by Student’s  t  test.  (D)  IHC staining of LDH5, GLUT1, PrlR and Stat5 with consecutive slides of a representative T47D xenograft tumor.  (E)  Co-staining of pYStat5 and GLUT1 in representative T47D xenografts from mice that were injected intraperitoneally with vehicle or human prolactin. Red, pYStat5; green, GLUT1. AQUA, automated quantitative analysis; GLUT1, glucose transporter 1; IHC, immunohistchemistry; LDH5, lactate dehydrogenase-5; Nuc-pYStat5, nuclear localized and tyrosine phosphorylated Stat5; PrlR, prolactin receptor; SD, standard deviation.

    Journal: Breast Cancer Research : BCR

    Article Title: Prolactin-Stat5 signaling in breast cancer is potently disrupted by acidosis within the tumor microenvironment

    doi: 10.1186/bcr3467

    Figure Lengend Snippet: Elevated GLUT1 is associated with low Nuc-pYStat5 in human breast cancer and xenografts. (A) Representative images from a breast cancer progression tissue array co-stained with anti-cytokeratins, DAPI, anti-pYStat5 or anti-GLUT1. (B) Expression of GLUT1 and nuclear pY-Stat5 in each tissue samples were quantified by AQUA and plotted. The range of normal GLUT1 expression is labeled on the plot by mean of normal mammary gland GLUT1 (log) score ± 3 SD. (C) Comparison of AQUA scores Nuc-pYStat5, Stat5a, Stat5b and PrlR between GLUT1 negative and positive breast cancer samples ( n  = 88) by Student’s t test. (D) IHC staining of LDH5, GLUT1, PrlR and Stat5 with consecutive slides of a representative T47D xenograft tumor. (E) Co-staining of pYStat5 and GLUT1 in representative T47D xenografts from mice that were injected intraperitoneally with vehicle or human prolactin. Red, pYStat5; green, GLUT1. AQUA, automated quantitative analysis; GLUT1, glucose transporter 1; IHC, immunohistchemistry; LDH5, lactate dehydrogenase-5; Nuc-pYStat5, nuclear localized and tyrosine phosphorylated Stat5; PrlR, prolactin receptor; SD, standard deviation.

    Article Snippet: Monoclonal antibodies used for immunohistochemistry include anti-pancytokeratin, anti-estrogen receptor (ER) (1D5), anti-progesterone receptor (PR) (PgR636) and antiKi67 (MIB-1) from Dako (Carpinteria, CA, USA), pY-Stat5 antibody (Epitomics, Burlingame, CA, USA), anti-glucose transporter 1 (GLUT1) from Thermo Fisher Scientific (Fremont, CA, USA), anti-PrlR (ECD) (1A2B1; Invitrogen, Carlsbad, CA, USA) and anti-lactate dehydrogenase-5 (LDH5) (Abcam, Cambridge, CA, USA).

    Techniques: Staining, Expressing, Labeling, Immunohistochemistry, Mouse Assay, Injection, Standard Deviation

    Mutually exclusive expression of nuclear-localized pYStat5 and GLUT1 in human breast cancer. (A)  Five human breast cancer cell lines were treated with prolactin or vehicle for 15 min at pH e  7.4 or pH e  6.8. Cell lysates were immunoprecipitated with anti-Stat5, resolved by SDS-PAGE and immunoblotted with anti-pYStat5 or anti-Stat5.  (B)  Median GLUT1 and Nuc-pYStat5 levels (AQUA scores) of the 52 breast cancer specimens in Cohort I are shown. Red dashed lines indicate the cutoff values for GLUT1 positivity and Nuc-pYStat5 positivity.  (C)  Example of a human invasive ductal carcinoma showing regional variability of pYStat5 IHC staining intensity.  (D)  GLUT1 and Nuc-pYStat5 AQUA scores of 2,244 randomly sampled tumor regions from 52 breast cancer specimens of Cohort 1. Examples of co-staining images show high GLUT1/low Nuc-pYStat5 (panel 1), high GLUT1/high Nuc-pYStat5 (panel 2), or low GLUT1/low Nuc-pYStat5 (Panel 3).  (E)  Cellular GLUT1 and Nuc-pYStat5 intensities in 8,804 cells sampled from six tumor spots displaying both high GLUT1 and high Nuc-pYStat5 are scatter-plotted. Representative images used for the analysis are shown. AQUA, automated quantitative analysis; GLUT1, glucose transporter 1; IHC, immunohistochemistry; Nuc-pYStat5, nuclear localized and tyrosine phosphorylated Stat5; pH e , extracellular pH.

    Journal: Breast Cancer Research : BCR

    Article Title: Prolactin-Stat5 signaling in breast cancer is potently disrupted by acidosis within the tumor microenvironment

    doi: 10.1186/bcr3467

    Figure Lengend Snippet: Mutually exclusive expression of nuclear-localized pYStat5 and GLUT1 in human breast cancer. (A) Five human breast cancer cell lines were treated with prolactin or vehicle for 15 min at pH e 7.4 or pH e 6.8. Cell lysates were immunoprecipitated with anti-Stat5, resolved by SDS-PAGE and immunoblotted with anti-pYStat5 or anti-Stat5. (B) Median GLUT1 and Nuc-pYStat5 levels (AQUA scores) of the 52 breast cancer specimens in Cohort I are shown. Red dashed lines indicate the cutoff values for GLUT1 positivity and Nuc-pYStat5 positivity. (C) Example of a human invasive ductal carcinoma showing regional variability of pYStat5 IHC staining intensity. (D) GLUT1 and Nuc-pYStat5 AQUA scores of 2,244 randomly sampled tumor regions from 52 breast cancer specimens of Cohort 1. Examples of co-staining images show high GLUT1/low Nuc-pYStat5 (panel 1), high GLUT1/high Nuc-pYStat5 (panel 2), or low GLUT1/low Nuc-pYStat5 (Panel 3). (E) Cellular GLUT1 and Nuc-pYStat5 intensities in 8,804 cells sampled from six tumor spots displaying both high GLUT1 and high Nuc-pYStat5 are scatter-plotted. Representative images used for the analysis are shown. AQUA, automated quantitative analysis; GLUT1, glucose transporter 1; IHC, immunohistochemistry; Nuc-pYStat5, nuclear localized and tyrosine phosphorylated Stat5; pH e , extracellular pH.

    Article Snippet: Monoclonal antibodies used for immunohistochemistry include anti-pancytokeratin, anti-estrogen receptor (ER) (1D5), anti-progesterone receptor (PR) (PgR636) and antiKi67 (MIB-1) from Dako (Carpinteria, CA, USA), pY-Stat5 antibody (Epitomics, Burlingame, CA, USA), anti-glucose transporter 1 (GLUT1) from Thermo Fisher Scientific (Fremont, CA, USA), anti-PrlR (ECD) (1A2B1; Invitrogen, Carlsbad, CA, USA) and anti-lactate dehydrogenase-5 (LDH5) (Abcam, Cambridge, CA, USA).

    Techniques: Expressing, Immunoprecipitation, SDS Page, Immunohistochemistry, Staining

    Effect of inhibiting the RNA futile cycle on the antibiotic tolerances of MazF persisters. E. coli MO:: mazE - mazF was diluted in Gutnick-glucose medium supplemented with aTc, cultured for 3 h, and treated with no inhibitor or Rif. One hour posttreatment, cells were exposed to no antibiotics (A), Amp (B), Ofl (C), Kan (D), or Strep (E). Rif treatment caused reduced culturability at time zero, which was regained during the course of the assay (refer to Fig. S6 in the supplemental material for supporting data). In MazF-accumulating cells, Rif treatment did not alter Amp or Ofl persister levels compared with the untreated control. However, MazF-accumulating cells were sensitive to Kan and Strep, and an increase in persister levels was observed only when transcription and, as a consequence, translation were inhibited with Rif treatment.

    Journal: mBio

    Article Title: RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

    doi: 10.1128/mBio.01588-15

    Figure Lengend Snippet: Effect of inhibiting the RNA futile cycle on the antibiotic tolerances of MazF persisters. E. coli MO:: mazE - mazF was diluted in Gutnick-glucose medium supplemented with aTc, cultured for 3 h, and treated with no inhibitor or Rif. One hour posttreatment, cells were exposed to no antibiotics (A), Amp (B), Ofl (C), Kan (D), or Strep (E). Rif treatment caused reduced culturability at time zero, which was regained during the course of the assay (refer to Fig. S6 in the supplemental material for supporting data). In MazF-accumulating cells, Rif treatment did not alter Amp or Ofl persister levels compared with the untreated control. However, MazF-accumulating cells were sensitive to Kan and Strep, and an increase in persister levels was observed only when transcription and, as a consequence, translation were inhibited with Rif treatment.

    Article Snippet: At specified time points or cell densities following dilution in Gutnick-glucose with aTc or aTc and Ara, 500-µl aliquots of culture were transferred into 17- by 100-mm polypropylene test tubes (Thermo, Fisher Scientific), treated with antibiotics, and incubated at 37°C with shaking at 250 rpm.

    Techniques: Cell Culture

    Quantification of oxygen and glucose consumption in MazF model persisters. E. coli MO:: mazE - mazF was grown to exponential phase before being washed and diluted in Gutnick with 2 mM glucose supplemented with aTc for MazF accumulation (red lines) or aTc with Ara for MazE-MazF coexpression (blue lines) at 0 h. (A) Growth of E. coli accumulating MazF or both MazE and MazF in the bioreactor where oxygen measurements were performed. (B) Oxygen consumption of cells accumulating MazF or expressing MazE-MazF measured over 5 h following inoculation in the bioreactor. The equilibrium oxygen concentration of the medium was 210 µM (dashed line). Cells undergoing MazF-mediated growth inhibition continued to consume oxygen, as illustrated by the concentration of dissolved oxygen in the growth medium remaining below 210 µM. (C) Glucose consumption of cells accumulating MazF or MazE-MazF. In the MazF samples, glucose consumption continued during growth inhibition. In the MazE-MazF sample, glucose exhaustion occurred around 4.5 h, and the cessation of glucose consumption coincided with an increase in dissolved oxygen in the growth medium. (D) Oxygen consumed per glucose consumed was calculated using data points from 2.5 to 5 h. Cells accumulating MazF consumed ~50% more oxygen per glucose compared with the MazE-MazF coexpression control. (E) Oxygen consumed relative to biomass produced was calculated using data points from 2.5 to 5 h, and it was 2.8-fold higher in MazF-inhibited cells than in cells expressing MazE-MazF. (F) Glucose consumed relative to biomass produced (calculated using data points from 2.5 to 5 h) was 1.9-fold higher than that of the coexpression control. *, P ≤ 0.05.

    Journal: mBio

    Article Title: RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

    doi: 10.1128/mBio.01588-15

    Figure Lengend Snippet: Quantification of oxygen and glucose consumption in MazF model persisters. E. coli MO:: mazE - mazF was grown to exponential phase before being washed and diluted in Gutnick with 2 mM glucose supplemented with aTc for MazF accumulation (red lines) or aTc with Ara for MazE-MazF coexpression (blue lines) at 0 h. (A) Growth of E. coli accumulating MazF or both MazE and MazF in the bioreactor where oxygen measurements were performed. (B) Oxygen consumption of cells accumulating MazF or expressing MazE-MazF measured over 5 h following inoculation in the bioreactor. The equilibrium oxygen concentration of the medium was 210 µM (dashed line). Cells undergoing MazF-mediated growth inhibition continued to consume oxygen, as illustrated by the concentration of dissolved oxygen in the growth medium remaining below 210 µM. (C) Glucose consumption of cells accumulating MazF or MazE-MazF. In the MazF samples, glucose consumption continued during growth inhibition. In the MazE-MazF sample, glucose exhaustion occurred around 4.5 h, and the cessation of glucose consumption coincided with an increase in dissolved oxygen in the growth medium. (D) Oxygen consumed per glucose consumed was calculated using data points from 2.5 to 5 h. Cells accumulating MazF consumed ~50% more oxygen per glucose compared with the MazE-MazF coexpression control. (E) Oxygen consumed relative to biomass produced was calculated using data points from 2.5 to 5 h, and it was 2.8-fold higher in MazF-inhibited cells than in cells expressing MazE-MazF. (F) Glucose consumed relative to biomass produced (calculated using data points from 2.5 to 5 h) was 1.9-fold higher than that of the coexpression control. *, P ≤ 0.05.

    Article Snippet: At specified time points or cell densities following dilution in Gutnick-glucose with aTc or aTc and Ara, 500-µl aliquots of culture were transferred into 17- by 100-mm polypropylene test tubes (Thermo, Fisher Scientific), treated with antibiotics, and incubated at 37°C with shaking at 250 rpm.

    Techniques: Acetylene Reduction Assay, Expressing, Concentration Assay, Inhibition, Produced

    Quantification of ribonucleotide pools in MazF model persisters during growth stasis. E. coli MO:: mazE and E. coli MO:: mazE - mazF were grown to exponential phase before being washed and diluted in Gutnick-glucose medium supplemented with aTc or aTc and Ara, which represents 0 h. Samples were collected and extracted 2, 3, and 4 h postdilution. Ratios of ribonucleotides in E. coli MO:: mazE and E. coli MO:: mazE - mazF were calculated by dividing the OD 600 -normalized abundances in aTc-induced cells by the OD 600 -normalized abundances in aTc and Ara-induced cells collected from the same experimental replicate. Averages and standard errors of the means (SEM) of ratios calculated from four experiments are shown. Ratios derived from E. coli MO:: mazE - mazF were compared with ratios derived from E. coli MO:: mazE (*, P ≤ 0.05). Overall, MazF accumulation resulted in statistically significant increases in all four ribonucleotide monophosphates. Greater relative increases in the abundances of NMPs and NDPs compared with their NTP counterparts are suggestive of low energy levels in MazF-accumulating cells.

    Journal: mBio

    Article Title: RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

    doi: 10.1128/mBio.01588-15

    Figure Lengend Snippet: Quantification of ribonucleotide pools in MazF model persisters during growth stasis. E. coli MO:: mazE and E. coli MO:: mazE - mazF were grown to exponential phase before being washed and diluted in Gutnick-glucose medium supplemented with aTc or aTc and Ara, which represents 0 h. Samples were collected and extracted 2, 3, and 4 h postdilution. Ratios of ribonucleotides in E. coli MO:: mazE and E. coli MO:: mazE - mazF were calculated by dividing the OD 600 -normalized abundances in aTc-induced cells by the OD 600 -normalized abundances in aTc and Ara-induced cells collected from the same experimental replicate. Averages and standard errors of the means (SEM) of ratios calculated from four experiments are shown. Ratios derived from E. coli MO:: mazE - mazF were compared with ratios derived from E. coli MO:: mazE (*, P ≤ 0.05). Overall, MazF accumulation resulted in statistically significant increases in all four ribonucleotide monophosphates. Greater relative increases in the abundances of NMPs and NDPs compared with their NTP counterparts are suggestive of low energy levels in MazF-accumulating cells.

    Article Snippet: At specified time points or cell densities following dilution in Gutnick-glucose with aTc or aTc and Ara, 500-µl aliquots of culture were transferred into 17- by 100-mm polypropylene test tubes (Thermo, Fisher Scientific), treated with antibiotics, and incubated at 37°C with shaking at 250 rpm.

    Techniques: Acetylene Reduction Assay, Derivative Assay

    Effects of transcription and translation inhibitors on the ribonucleotide pools of MazF persisters. Three hours after E. coli MO:: mazE - mazF was diluted and cultured in Gutnick-glucose with aTc to induce MazF accumulation (designated time zero), the samples were treated with no inhibitor, Rif, or Spec. Samples were then collected hourly for 2 h for LC-MS analysis. Ratios of ribonucleotides in inhibitor-treated samples to those in untreated samples were calculated by dividing the OD 600 -normalized abundances in treated samples by the normalized abundances in untreated samples collected from the same experimental replicate. Averages and SEM of ratios calculated from four experiments are shown. A ratio of 1, indicating no difference between the two samples, is depicted by the dashed line on each graph, and asterisks indicates statistically significant deviations from a value of 1 ( P ≤ 0.05). With the exception of CMP, Rif treatment decreased the abundances of NMPs. In contrast, the abundances of all four NTPs increased. These results suggested that Rif treatment led to the inhibition of the RNA futile cycle, which consequently increased the energy level of cells.

    Journal: mBio

    Article Title: RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

    doi: 10.1128/mBio.01588-15

    Figure Lengend Snippet: Effects of transcription and translation inhibitors on the ribonucleotide pools of MazF persisters. Three hours after E. coli MO:: mazE - mazF was diluted and cultured in Gutnick-glucose with aTc to induce MazF accumulation (designated time zero), the samples were treated with no inhibitor, Rif, or Spec. Samples were then collected hourly for 2 h for LC-MS analysis. Ratios of ribonucleotides in inhibitor-treated samples to those in untreated samples were calculated by dividing the OD 600 -normalized abundances in treated samples by the normalized abundances in untreated samples collected from the same experimental replicate. Averages and SEM of ratios calculated from four experiments are shown. A ratio of 1, indicating no difference between the two samples, is depicted by the dashed line on each graph, and asterisks indicates statistically significant deviations from a value of 1 ( P ≤ 0.05). With the exception of CMP, Rif treatment decreased the abundances of NMPs. In contrast, the abundances of all four NTPs increased. These results suggested that Rif treatment led to the inhibition of the RNA futile cycle, which consequently increased the energy level of cells.

    Article Snippet: At specified time points or cell densities following dilution in Gutnick-glucose with aTc or aTc and Ara, 500-µl aliquots of culture were transferred into 17- by 100-mm polypropylene test tubes (Thermo, Fisher Scientific), treated with antibiotics, and incubated at 37°C with shaking at 250 rpm.

    Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Inhibition

    Effect of transcription and translation inhibitors on oxygen and glucose consumption in E. coli accumulating MazF. E. coli MO:: mazE-mazF was grown to exponential phase and then pelleted, washed, and diluted in Gutnick medium with 2 mM glucose and aTc to induce MazF accumulation at 0 h. Dissolved oxygen and glucose consumption in the culture were measured in a bioreactor over 5 h. At 3 h postinoculation (red dashed line), inhibitors (100 µg/ml Rif, 100 µg/ml Spec, or 5 mM KCN) were added to each culture. Top panels compare oxygen consumption and bottom panels compare glucose consumption of untreated samples with those treated with Rif (A and D), KCN (B and E), or Spec (C and F). Inhibition of transcription in MazF-accumulating cells resulted in rapid decrease in oxygen consumption, which was similar to results with cells treated with the respiratory inhibitor KCN. Glucose consumption modestly and transiently decreased only in the Rif-treated sample, whereas cells treated with Spec or KCN exhibited glucose uptake similar to that in the untreated sample. *, P ≤ 0.05.

    Journal: mBio

    Article Title: RNA Futile Cycling in Model Persisters Derived from MazF Accumulation

    doi: 10.1128/mBio.01588-15

    Figure Lengend Snippet: Effect of transcription and translation inhibitors on oxygen and glucose consumption in E. coli accumulating MazF. E. coli MO:: mazE-mazF was grown to exponential phase and then pelleted, washed, and diluted in Gutnick medium with 2 mM glucose and aTc to induce MazF accumulation at 0 h. Dissolved oxygen and glucose consumption in the culture were measured in a bioreactor over 5 h. At 3 h postinoculation (red dashed line), inhibitors (100 µg/ml Rif, 100 µg/ml Spec, or 5 mM KCN) were added to each culture. Top panels compare oxygen consumption and bottom panels compare glucose consumption of untreated samples with those treated with Rif (A and D), KCN (B and E), or Spec (C and F). Inhibition of transcription in MazF-accumulating cells resulted in rapid decrease in oxygen consumption, which was similar to results with cells treated with the respiratory inhibitor KCN. Glucose consumption modestly and transiently decreased only in the Rif-treated sample, whereas cells treated with Spec or KCN exhibited glucose uptake similar to that in the untreated sample. *, P ≤ 0.05.

    Article Snippet: At specified time points or cell densities following dilution in Gutnick-glucose with aTc or aTc and Ara, 500-µl aliquots of culture were transferred into 17- by 100-mm polypropylene test tubes (Thermo, Fisher Scientific), treated with antibiotics, and incubated at 37°C with shaking at 250 rpm.

    Techniques: Inhibition

    Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Concentration Assay, Activity Assay

    Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Sequencing, Binding Assay, SPR Assay

    Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Mass Spectrometry, Incubation, Cell Culture, Alamar Blue Assay

    Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Crystal Violet Assay, Mass Spectrometry, Cell Culture, Incubation