Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans methods
Aim. Evidence-backed execution summary for Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans methods from Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans.
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mouse
Subject model for the experiment.
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Effects of blueberry polyphenols on intrinsic stress resistance
reagent used in the protocol.
- Use
- Interestingly, BB treatment did not improve survival under mild to severe oxidative stress. Resistance to oxidative stress was examined by exposing animals to hydrogen peroxide or paraquat, an intracellular free-radical-generating compound. BB treatment was not associated with any increase in survival in the presenc...
Experimental procedures
reagent used in the protocol.
- Use
- All strains were maintained at 15 °C on nematode growth medium (NGM) as described ( ). Strains used in this study were: N2, bristol (wild-type); BA17, fem-1(hc17); GR1307, daf-16(mgDf50); TK22, mev-1(kn1); VC199, sir-2.1(ok434); EU1, skn-1(zu67)/nT1; AM1, osr-1(rm1); AU1, sek-1(ag1); and MT2605, unc-43(n...
Phenotypic assays
reagent used in the protocol.
- Use
- For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the...
Phenotypic assays
reagent used in the protocol.
- Use
- Thermotolerance assays were performed with hermaphrodites on adult day 5, after the majority of egg-laying had ceased. Animals were transferred onto 3-cm NGM agar plates supplemented as indicated and then incubated at 35 °C for 16 h. Survival was scored as the number of animals responsive to gentle touch as a f...
Phenotypic assays
reagent used in the protocol.
- Use
- To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24...
Analysis of gene expression by reverse transcriptase PCR
reagent used in the protocol.
- Use
- For reverse transcriptase PCR (RT-PCR) analysis of gene expression, fem-1(hc17) animals were grown at an approximate density of 200 animals/plate in the presence or absence of 200 µg mL -1 BB polyphenols. Animals were kept at 25 °C except for heat-stressed samples, which were transferred to 35 °...
Phenotypic assays
For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the...
- Use
- For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the...
Phenotypic assays
To determine lipofuscin levels, adult hermaphrodites were anesthetized in 0.2% sodium azide and mounted on 2% agarose pads for visualization of intestinal fluorescence on a Nikon E800 microscope using an Endow GFP filter with a mercury UV source (Nikon). Images were captured using a constant exposure time with a Ham...
- Use
- To determine lipofuscin levels, adult hermaphrodites were anesthetized in 0.2% sodium azide and mounted on 2% agarose pads for visualization of intestinal fluorescence on a Nikon E800 microscope using an Endow GFP filter with a mercury UV source (Nikon). Images were captured using a constant exposure time with a Ham...
Phenotypic assays
To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24...
- Use
- To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24...
Phenotypic assays
Software used for acquisition, scoring, statistics, or reporting.
- Use
- For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the...
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Proanthocyanidin components of blueberries enhance longevity
Blueberries contain a mixture of different polyphenol compounds that can be separated into three primary fractions enriched in either anthocyanins (ATC), proanthocyanidins (PAC) or hydroxycinnamic esters, mainly chlorogenic acid (CA). Major components of each of these fractions have been shown to confer significant antioxidant activity and ATC can protect cells against oxidative stress in vitro (; ). To determine which fraction(s) delayed aging, we assayed their effects on C. elegans lifespan. Neither the ATC-enriched fraction nor purified CA had any significant effect on longevity ( ) (control, 12.0 ± 0.34 days; ATC, 11.7 ± 0.36 days, P = 0.96 vs. control; CA, 11.7 ± 0.39 days, P = 0.99). However, treatment with the PAC-enriched fraction increased lifespan to a similar extent as the starting BB polyphenol mixture or the remixed fractions ( ) (PAC, 14.4 ± 0.36 day...
Blueberry polyphenols do not appear to prolong lifespan through antimicrobial effects
One contributor to late-age mortality in C. elegans is the detrimental effect of the bacterial food source (; ). Accordingly, C. elegans lifespan could be increased approximately 30% when bacterial growth was arrested by ampicillin ( ). We observed that lifespan of BB-treated wild-type animals was not further extended by the presence of ampicillin to arrest bacterial growth ( ), indicating that BB may have extended lifespan by relieving microbial stress. To investigate this possibility, we first tested whether the BB polyphenols that prolong lifespan inhibited bacterial growth. We monitored growth of the bacterial lawn under conditions identical to C. elegans aging assays, 11 days on NGM agar at 25 °C, in the presence or absence of BB. During this period, untreated bacterial lawns completed approximately one population doubling, while bacterial growth was arrested after ampicill...
Blueberry polyphenols do not appear to prolong lifespan through antimicrobial effects
BB and antimicrobial treatment had different effects on Caenorhabditis elegans thermotolerance and lifespan. (A)BB treatment did not inhibit bacterial growth in aging assays. Bacterial lawn growth was monitored over 11 days under conditions replicating those of C. elegans aging assays. Shown are the average number of colony forming units (cfu) per lawn after indicated treatments in two trials, ** P < 0.01. (B) Effects of blueberry polyphenols and ampicillin on C. elegans thermotolerance measured as survivorship at 35 °C after 16 h, as in. Ampicillin treatment alone had no effect on thermotolerance. Shown are average survivorships in three trials, with at least 20 animals per trial, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) BB polyphenols (200 µg mL -1 ) and ampicillin had additive effects on daf-16( mgDf50) lifespan at 25 °C (purple curve). daf-16( mgDf50)...
Phenotypic assays
For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the nonpermissive temperature for fertility of fem-1(hc17). Lifespan scoring was initiated after hermaphrodites completed the final larval molt, on the first day of adulthood. For aging assays with BB extracts, treatments were added to NGM agar plates on the first day of the lifespan assay. For lifespan assays with fertile strains, hermaphrodites were transferred daily for the first 4 days of adulthood to avoid progeny overgrowth. In these cases, all treatment plates were prepared on day 0 of adulthood. Statistical analyses and survival plots of lifespan data were performed w...
Phenotypic assays
To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24 °C, washed and incubated overnight at 4 °C with Alexafluor-546-conjugated goat anti-mouse secondary antibody (1: 100) (#A-11003, Invitrogen, Carlsbad, CA, USA). Stained animals were mounted on 2% agarose pads and fluorescence visualized as for lipofuscin with appropriate filter sets. 4-HNE immunofluorescence was measured in the pharynx terminal bulb and somatic gonad using ImageJ software.
Measurement outputs
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We next examined the effects of BB treatment on a transcriptional marker of aging. Several independent analyses of gene expression during aging in C. elegans have revealed that...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
BB polyphenols reduced aging-related increase of inducible hsp transcripts. Expression levels of small heat-shock proteins, relative to actin, were determined by RT-PCR in popul...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We next considered the possibility that BB treatment acted as a mild stressor that induced expression of protective enzymes and this increased gene expression delayed aging ( )....
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In C. elegans, two transcription factors, DAF-16 and SKN-1, promote expression of antioxidant or detoxification enzymes. The DAF-16/FOXO transcription factor promotes expressio...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
BB polyphenols reduced aging-related increase of inducible hsp transcripts.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We next examined the effects of BB treatment on a transcriptional marker of aging. Several independent analyses of gene expression during aging in C. elegans have revealed that...; BB polyphenols reduced aging-related increase of inducible hsp transcripts. Expression levels of small heat-shock proteins, relative to actin, were determined by RT-PCR in popul...; We next considered the possibility that BB treatment acted as a mild stressor that induced expression of protective enzymes and this increased gene expression delayed aging ( )....; In C. elegans, two transcription factors, DAF-16 and SKN-1, promote expression of antioxidant or detoxification enzymes. The DAF-16/FOXO transcription factor promotes expressio....
from paperStatistical comparison
BB polyphenols reduced aging-related increase of inducible hsp transcripts. Expression levels of small heat-shock proteins, relative to actin, were determined by RT-PCR in popul...; Blueberries contain a mixture of different polyphenol compounds that can be separated into three primary fractions enriched in either anthocyanins (ATC), proanthocyanidins (PAC)...; A PAC-enriched fraction of BB contained components sufficient to extend Caenorhabditis elegans lifespan. The total blueberry polyphenols were fractionated by C18 and Sephadex LH...; Treatment with blueberry polyphenols improved thermotolerance, but not oxidative stress resistance. (A)Fractional survival at 35 °C for day 5 fem-1 ( hc17) or animals with...
from paperReporting output
Report representative outputs alongside summary comparisons for We next examined the effects of BB treatment on a transcriptional marker of aging. Several independent analyses of gene expression during aging in C. elegans have revealed that..., BB polyphenols reduced aging-related increase of inducible hsp transcripts. Expression levels of small heat-shock proteins, relative to actin, were determined by RT-PCR in popul..., We next considered the possibility that BB treatment acted as a mild stressor that induced expression of protective enzymes and this increased gene expression delayed aging ( )...., In C. elegans, two transcription factors, DAF-16 and SKN-1, promote expression of antioxidant or detoxification enzymes. The DAF-16/FOXO transcription factor promotes expressio....
inferred from protocolStructured statistical methods
BB polyphenols reduced aging-related increase of inducible hsp transcripts. Expression levels of small heat-shock proteins, relative to actin, were determined by RT-PCR in popul...; Blueberries contain a mixture of different polyphenol compounds that can be separated into three primary fractions enriched in either anthocyanins (ATC), proanthocyanidins (PAC)...; A PAC-enriched fraction of BB contained components sufficient to extend Caenorhabditis elegans lifespan. The total blueberry polyphenols were fractionated by C18 and Sephadex LH...; Treatment with blueberry polyphenols improved thermotolerance, but not oxidative stress resistance. (A)Fractional survival at 35 °C for day 5 fem-1 ( hc17) or animals with...
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Evidence quotes (5)
Blueberries contain a mixture of different polyphenol compounds that can be separated into three primary fractions enriched in either anthocyanins (ATC), proanthocyanidins (PAC) or hydroxycinnamic esters, mainly chlorogenic acid (CA). Major components of each of these fractions have been shown to confer significant antioxidant activity and ATC can protect cells against oxidative stress in vitro (; ). To determine which fraction(s) delayed aging, we assayed their effects on C. elegans lifespan. Neither the ATC-enriched fraction nor purified CA had any significant effect on longevity ( ) (control, 12.0 ± 0.34 days; ATC, 11.7 ± 0.36 days, P = 0.96 vs. control; CA, 11.7 ± 0.39 days, P = 0.99). However, treatment with the PAC-enriched fraction increased lifespan to a similar extent as the starting BB polyphenol mixture or the remixed fractions ( ) (PAC, 14.4 ± 0.36 days, P < 0.0001 vs. control; start, 14.8 ± 0.36 days, P < 0.0001; remix, 14.0 ± 0.48 days, P < 0.0001; complete statistics for lifespan trials with PAC-enriched fraction are presented in ). Thus, components of the PAC-enriched fraction of blueberries could extend lifespan in C. elegans.
One contributor to late-age mortality in C. elegans is the detrimental effect of the bacterial food source (; ). Accordingly, C. elegans lifespan could be increased approximately 30% when bacterial growth was arrested by ampicillin ( ). We observed that lifespan of BB-treated wild-type animals was not further extended by the presence of ampicillin to arrest bacterial growth ( ), indicating that BB may have extended lifespan by relieving microbial stress. To investigate this possibility, we first tested whether the BB polyphenols that prolong lifespan inhibited bacterial growth. We monitored growth of the bacterial lawn under conditions identical to C. elegans aging assays, 11 days on NGM agar at 25 °C, in the presence or absence of BB. During this period, untreated bacterial lawns completed approximately one population doubling, while bacterial growth was arrested after ampicillin treatment. In contrast, BB polyphenols had no effect on bacterial population growth at the doses tested in lifespan assays ( ). Furthermore, C. elegans nematodes grown on ampicillin-treated bacteria displayed no increase in thermotolerance, nor did the combination of ampicillin and BB together en...
BB and antimicrobial treatment had different effects on Caenorhabditis elegans thermotolerance and lifespan. (A)BB treatment did not inhibit bacterial growth in aging assays. Bacterial lawn growth was monitored over 11 days under conditions replicating those of C. elegans aging assays. Shown are the average number of colony forming units (cfu) per lawn after indicated treatments in two trials, ** P < 0.01. (B) Effects of blueberry polyphenols and ampicillin on C. elegans thermotolerance measured as survivorship at 35 °C after 16 h, as in. Ampicillin treatment alone had no effect on thermotolerance. Shown are average survivorships in three trials, with at least 20 animals per trial, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) BB polyphenols (200 µg mL -1 ) and ampicillin had additive effects on daf-16( mgDf50) lifespan at 25 °C (purple curve). daf-16( mgDf50), untreated control, mean 11.6 days, 0.28 SE, n = 69; 200 µg mL -1 BB polyphenols, mean 13.0 days, 0.27 SE, n = 49; 100 µg mL -1 ampicillin, mean 12.7 days, 0.35 SE, n = 53; ampicillin + BB polyphenols, mean 14.7 days, 0.33 SE, n = 60; P < 0.0001, log-rank, for Amp. vs. BB+Amp...
For aging assays, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the nonpermissive temperature for fertility of fem-1(hc17). Lifespan scoring was initiated after hermaphrodites completed the final larval molt, on the first day of adulthood. For aging assays with BB extracts, treatments were added to NGM agar plates on the first day of the lifespan assay. For lifespan assays with fertile strains, hermaphrodites were transferred daily for the first 4 days of adulthood to avoid progeny overgrowth. In these cases, all treatment plates were prepared on day 0 of adulthood. Statistical analyses and survival plots of lifespan data were performed with JMP analysis software (SAS Institute Inc, Cary, NC, USA). Pharynx pumping rates were scored on adults at room temperature (24 °C) under a Nikon SMZ1500 stereomicroscope (Nikon, Melville, NY, USA).
To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described ( ). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1: 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24 °C, washed and incubated overnight at 4 °C with Alexafluor-546-conjugated goat anti-mouse secondary antibody (1: 100) (#A-11003, Invitrogen, Carlsbad, CA, USA). Stained animals were mounted on 2% agarose pads and fluorescence visualized as for lipofuscin with appropriate filter sets. 4-HNE immunofluorescence was measured in the pharynx terminal bulb and somatic gonad using ImageJ software.
Machine-readable layer
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