Structural modulation of gut microbiota in life-long calorie-restricted mice methods
Aim. Evidence-backed execution summary for Structural modulation of gut microbiota in life-long calorie-restricted mice methods from Structural modulation of gut microbiota in life-long calorie-restricted mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
CR reduces antigen load to the hosts from the gut microbiota
reagent used in the protocol.
- Use
- Through long-term CR, the relative abundance of the OTUs negatively correlated with lifespan was significantly reduced, and the relative abundance of OTUs positively correlated with lifespan was increased in mice both on low-fat diet and high-fat diet ( and ). The serum levels of lipopolysaccharide (LPS)-binding pro...
Methods
reagent used in the protocol.
- Use
- The animal experimental procedures, approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, CAS, were described previously by Zhou et al. Male C57BL/6 J mice at 5 weeks of age were randomly assigned to one of the six groups ( n =30 for each group) and individually cage...
Methods
reagent used in the protocol.
- Use
- Fresh faecal matter was collected from the above animal trial at 62 weeks (LFD, n =21; LFD+CR, n =29; LFD+Ex, n =22; HFD, n =28; HFD+CR, n =29; and HFD+Ex, n =23), 83 weeks (LFD, n =16; LFD+CR, n =22; LFD+Ex, n =19; HFD, n =12; HFD+CR, n =14; and HFD+Ex, n =15) and 141 weeks (LFD, n =6; LFD+CR, n =15; LFD+Ex, n =6;...
Pyrosequencing of the V3 region of 16S rRNA genes
reagent used in the protocol.
- Use
- DNA was extracted using the PSP®Spin Stool DNA Plus Kit (Invitek GmbH, Germany). The primers P1 and P2 (5′-NNNNNN CCTACGGGAGGCAGCAG -3′ and 5′-NNNNNN ATTACCGCGGCTGCT -3′) correspond to positions 341 to 534 in the Escherichia coli 16S rRNA gene, with a sample-unique DNA barcode of six-mer...
Bioinformatics and statistical analysis of sequencing data
reagent used in the protocol.
- Use
- The standards for quality control of selecting valid reads for analysis were as follows: if a sequence (a) shows no mismatch to the barcode and 16S rRNA gene primer at sequencing end, (b) is more than 100 nucleotides in length, (c) has no more than two undermined bases in the sequence read and (d) finds >75% mach to...
Serum LBP measurements
reagent used in the protocol.
- Use
- Blood samples were collected from the tail vein after overnight fasting and centrifuged at 12,000 r.p.m. for 30 min to pellet blood cells, and the serum was stored at -80 °C until further analyses. Serum LBP was determined after a dilution of 1:800 using the Mouse Lipopolysaccharide Bind...
Pyrosequencing of the V3 region of 16S rRNA genes
DNA was extracted using the PSP®Spin Stool DNA Plus Kit (Invitek GmbH, Germany). The primers P1 and P2 (5′-NNNNNN CCTACGGGAGGCAGCAG -3′ and 5′-NNNNNN ATTACCGCGGCTGCT -3′) correspond to positions 341 to 534 in the Escherichia coli 16S rRNA gene, with a sample-unique DNA barcode of six-mer...
- Use
- DNA was extracted using the PSP®Spin Stool DNA Plus Kit (Invitek GmbH, Germany). The primers P1 and P2 (5′-NNNNNN CCTACGGGAGGCAGCAG -3′ and 5′-NNNNNN ATTACCGCGGCTGCT -3′) correspond to positions 341 to 534 in the Escherichia coli 16S rRNA gene, with a sample-unique DNA barcode of six-mer...
Bioinformatics and statistical analysis of sequencing data
The standards for quality control of selecting valid reads for analysis were as follows: if a sequence (a) shows no mismatch to the barcode and 16S rRNA gene primer at sequencing end, (b) is more than 100 nucleotides in length, (c) has no more than two undermined bases in the sequence read and (d) finds >75% mach to...
- Use
- The standards for quality control of selecting valid reads for analysis were as follows: if a sequence (a) shows no mismatch to the barcode and 16S rRNA gene primer at sequencing end, (b) is more than 100 nucleotides in length, (c) has no more than two undermined bases in the sequence read and (d) finds >75% mach to...
Bioinformatics and statistical analysis of sequencing data
LEfSe is an algorithm for high-dimensional biomarker discovery and explanation that identifies genomic features (genes, pathways or taxa) characterizing the differences between two or more biological conditions (or classes; see figure below). LEfSe emphasizes both statistical significance and biological relevance, a...
- Use
- LEfSe is an algorithm for high-dimensional biomarker discovery and explanation that identifies genomic features (genes, pathways or taxa) characterizing the differences between two or more biological conditions (or classes; see figure below). LEfSe emphasizes both statistical significance and biological relevance, a...
Bioinformatics and statistical analysis of sequencing data
Based on two diets, associations between each OTU (filtered for an OTU subject prevalence of at least 10%) at 62 weeks and lifespan were determined using the Kendall tau rank correlation coefficient under Matlab (ver. 7.1; The MathWorks, Inc.). The OTU was considered significantly correlated with lifespan for P <0.0...
- Use
- Based on two diets, associations between each OTU (filtered for an OTU subject prevalence of at least 10%) at 62 weeks and lifespan were determined using the Kendall tau rank correlation coefficient under Matlab (ver. 7.1; The MathWorks, Inc.). The OTU was considered significantly correlated with lifespan for P <0.0...
Bioinformatics and statistical analysis of sequencing data
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The standards for quality control of selecting valid reads for analysis were as follows: if a sequence (a) shows no mismatch to the barcode and 16S rRNA gene primer at sequencing end, (b) is more than 100 nucleotides in length, (c) has no more than two undermined bases in the sequence read and (d) finds >75% mach to...
Bioinformatics and statistical analysis of sequencing data
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Based on two diets, associations between each OTU (filtered for an OTU subject prevalence of at least 10%) at 62 weeks and lifespan were determined using the Kendall tau rank correlation coefficient under Matlab (ver. 7.1; The MathWorks, Inc.). The OTU was considered significantly correlated with lifespan for P <0.0...
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Specific phylotypes modulated by life-long CR
As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to identify specific phylotypes responding to life-long CR at both mid-life (62 weeks of age) and late life (141 weeks of age). We did not analyse data at 83 weeks of age because there is no significant age-related shift of gut microbiota between 62 and 83 weeks.
Specific phylotypes modulated by life-long CR
At the late-life age of 141 weeks, 27 OTUs were higher and 27 were lower in the LFD+CR group than in the LFD group ( and ). Ten of these OTUs were also significantly different between the two treatment groups in mid-life. For example, although OTU45 in the genus Lactobacillus was not the predominant phylotype in bacterial communities of LFD+CR mice, the relative abundance of this OTU was still higher in LFD+CR mice than in LFD mice (1.7% versus 0.024%, respectively; P <0.001, one-way ANOVA). Different from mid-life, the OTUs belonging to Bifidobacterium were higher in LFD+CR mice, but the OTU469 of Desulfovibrionaceae was lower in LFD+CR mice. Some members in the genus Bifidobacterium are well-known probiotic strains, and some in the family Desulfovibrionaceae have previously been found to be positively associated with obesity and inflammation.
Specific phylotypes modulated by life-long CR
The mice had a significantly different gut microbiota structure between the LFD and HFD groups ( ), confirming results of previous studies. CR also shifted the gut microbiota in mice fed with high-fat diet but not as dramatic compared with their low-fat diet companions ( and ). In mid-life, 30 phylotypes were selected as key variables for separating the gut microbiota under different food intake conditions ( and ); 18 of them were higher and 12 were lower in the HFD+CR group than in the HFD group. All the phylotypes in Porphyromonadaceae were higher in the HFD+CR than in the HFD group. Most of the OTUs responding to CR in HFD+CR mice were not found in LFD+CR mice. Only three OTUs (in Lactococcus (OTU65), Bacteroidales (OTU366) and Peptostreptococcaceae (OTU37), respectively) were reduced, and three OTUs in Tannerella (OTU119, 155 and 267) were increased by CR both with mice fed with...
Structural modulation of gut microbiota during aging
Further analysis suggested that CR significantly affected the succession of gut microbiota during aging. Division-level analysis showed that the Firmicutes / Bacteroidetes ratio of gut microbiota in all the mouse groups decreased from 62 to 141 weeks ( ). Using LEfSe, we compared the gut microbiota of mice in each group between mid-life and late life to identify the specific phylotypes with OTU levels associated with aging.
Structural modulation of gut microbiota during aging
In LFD mice, the phylotypes mainly responsible for decrease of the phylum Firmicutes during aging were in the genus Allobaculum (12 OTUs; 28.1% at 62 weeks versus 0.20% at 141 weeks) ( and ). In LFD+CR mice, not only OTUs in the genus Allobaculum (29.7% at 62 weeks versus 7.2% at 141 weeks) but also OTUs in the genus Lactobacillus (21.0% at 62 weeks versus 2.0% at 141 weeks) made a significant contribution to the decrease of Firmicutes during aging ( and ).
Structural modulation of gut microbiota during aging
Conversely, the increase of the phylum Bacteroidetes during aging in LFD mice was due to the increase of OTUs in the genus Bacteroides (family Bacteroidaceae; 0.84% at 62 weeks versus 26.7% at 141 weeks). However, in LFD+CR mice, OTUs in the family Porphyromonadeceae (9.4% at 62 weeks versus 28.2% at 141 weeks), instead of bacteria in Bacteroides, were largely responsible for the increase of Bacteroidetes with age. Thus, the apparent phylum level changes associated with aging were actually mediated by different phylogenetic groups in animals with or without CR treatment.
Correlation of mid-life gut microbiota with lifespan
We next used the Kendall tau rank correlation coefficient to directly measure the correlation between the phylotypes of gut microbiota in mid-life and the lifespan based on two types of diet. We identified that 45 OTUs significantly correlated with lifespan in mice fed on low-fat diet. Except for one Bacteroidales OTU, the remaining 15 OTUs significantly positively correlated with lifespan belonged to Firmicutes. Particularly, eight OTUs in Lactobacillus showed strong correlation with lifespan. The 30 phylotypes negatively correlated with lifespan were distributed in the five Phyla of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and TM7 ( and ). In the mice on the high-fat diet, 20 OTUs were positively correlated, and 18 OTUs were negatively correlated with lifespan, most of which were in Firmicutes and Bacteroidetes, except for two Actinobacteria OTUs ( and ). Becau...
Methods
The animal experimental procedures, approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, CAS, were described previously by Zhou et al. Male C57BL/6 J mice at 5 weeks of age were randomly assigned to one of the six groups ( n =30 for each group) and individually caged for a life-long trial: (1) low-fat diet with sedentary activity (LFD), (2) low-fat diet with 30% CR and sedentary activity (LFD+CR), (3) low-fat diet with voluntary running exercise (LFD+Ex), (4) high-fat diet with sedentary activity (HFD), (5) high-fat die with 30% CR and sedentary activity (HFD+CR) and (6) high-fat diet with voluntary running exercise (HFD+Ex). All faecal and serum samples for the current study were collected from this animal trial.
Measurement outputs
What raw and processed outputs should exist?
We also employed partial least square discriminate analysis to confirm the results, and the identified specific phylotypes responding to life-long CR were similar to those from...
- 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
As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to i...
- 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 also identified a few OTUs that were different between the mice in the exercise group and their ad libitum companions by LEfSe; however, the relative abundance of these OTUs...
- 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
Further analysis suggested that CR significantly affected the succession of gut microbiota during aging. Division-level analysis showed that the Firmicutes / Bacteroidetes ratio...
- 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
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to identify specific phylotypes responding to life-long CR at both mid-life (62 weeks of age) and late l...
from paperScoring or quantification
Quantify the primary readouts for this experiment: We also employed partial least square discriminate analysis to confirm the results, and the identified specific phylotypes responding to life-long CR were similar to those from...; As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to i...; We also identified a few OTUs that were different between the mice in the exercise group and their ad libitum companions by LEfSe; however, the relative abundance of these OTUs...; Further analysis suggested that CR significantly affected the succession of gut microbiota during aging. Division-level analysis showed that the Firmicutes / Bacteroidetes ratio....
from paperStatistical comparison
As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to i...; In mid-life, 34 phylotypes at the OTU level were discovered as high-dimensional biomarkers for separating gut microbiota between LFD and LFD+CR mice ( and ). Sixteen of these OT...; At the late-life age of 141 weeks, 27 OTUs were higher and 27 were lower in the LFD+CR group than in the LFD group ( and ). Ten of these OTUs were also significantly different b...; We also identified a few OTUs that were different between the mice in the exercise group and their ad libitum companions by LEfSe; however, the relative abundance of these OTUs...
from paperReporting output
Report representative outputs alongside summary comparisons for We also employed partial least square discriminate analysis to confirm the results, and the identified specific phylotypes responding to life-long CR were similar to those from..., As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to i..., We also identified a few OTUs that were different between the mice in the exercise group and their ad libitum companions by LEfSe; however, the relative abundance of these OTUs..., Further analysis suggested that CR significantly affected the succession of gut microbiota during aging. Division-level analysis showed that the Firmicutes / Bacteroidetes ratio....
inferred from protocolStructured statistical methods
As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to i...; In mid-life, 34 phylotypes at the OTU level were discovered as high-dimensional biomarkers for separating gut microbiota between LFD and LFD+CR mice ( and ). Sixteen of these OT...; At the late-life age of 141 weeks, 27 OTUs were higher and 27 were lower in the LFD+CR group than in the LFD group ( and ). Ten of these OTUs were also significantly different b...; We also identified a few OTUs that were different between the mice in the exercise group and their ad libitum companions by LEfSe; however, the relative abundance of these OTUs...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) was employed to identify specific phylotypes responding to life-long CR at both mid-life (62 weeks of age) and late life (141 weeks of age). We did not analyse data at 83 weeks of age because there is no significant age-related shift of gut microbiota between 62 and 83 weeks.
At the late-life age of 141 weeks, 27 OTUs were higher and 27 were lower in the LFD+CR group than in the LFD group ( and ). Ten of these OTUs were also significantly different between the two treatment groups in mid-life. For example, although OTU45 in the genus Lactobacillus was not the predominant phylotype in bacterial communities of LFD+CR mice, the relative abundance of this OTU was still higher in LFD+CR mice than in LFD mice (1.7% versus 0.024%, respectively; P <0.001, one-way ANOVA). Different from mid-life, the OTUs belonging to Bifidobacterium were higher in LFD+CR mice, but the OTU469 of Desulfovibrionaceae was lower in LFD+CR mice. Some members in the genus Bifidobacterium are well-known probiotic strains, and some in the family Desulfovibrionaceae have previously been found to be positively associated with obesity and inflammation.
The mice had a significantly different gut microbiota structure between the LFD and HFD groups ( ), confirming results of previous studies. CR also shifted the gut microbiota in mice fed with high-fat diet but not as dramatic compared with their low-fat diet companions ( and ). In mid-life, 30 phylotypes were selected as key variables for separating the gut microbiota under different food intake conditions ( and ); 18 of them were higher and 12 were lower in the HFD+CR group than in the HFD group. All the phylotypes in Porphyromonadaceae were higher in the HFD+CR than in the HFD group. Most of the OTUs responding to CR in HFD+CR mice were not found in LFD+CR mice. Only three OTUs (in Lactococcus (OTU65), Bacteroidales (OTU366) and Peptostreptococcaceae (OTU37), respectively) were reduced, and three OTUs in Tannerella (OTU119, 155 and 267) were increased by CR both with mice fed with high-fat diet and low-fat diet. Because all the HFD mice had died before 141 weeks, we could not obtain any data regarding gut microbiota responding to restriction of high-fat diet intake at the late-life stage of mice.
Further analysis suggested that CR significantly affected the succession of gut microbiota during aging. Division-level analysis showed that the Firmicutes / Bacteroidetes ratio of gut microbiota in all the mouse groups decreased from 62 to 141 weeks ( ). Using LEfSe, we compared the gut microbiota of mice in each group between mid-life and late life to identify the specific phylotypes with OTU levels associated with aging.
In LFD mice, the phylotypes mainly responsible for decrease of the phylum Firmicutes during aging were in the genus Allobaculum (12 OTUs; 28.1% at 62 weeks versus 0.20% at 141 weeks) ( and ). In LFD+CR mice, not only OTUs in the genus Allobaculum (29.7% at 62 weeks versus 7.2% at 141 weeks) but also OTUs in the genus Lactobacillus (21.0% at 62 weeks versus 2.0% at 141 weeks) made a significant contribution to the decrease of Firmicutes during aging ( and ).
Conversely, the increase of the phylum Bacteroidetes during aging in LFD mice was due to the increase of OTUs in the genus Bacteroides (family Bacteroidaceae; 0.84% at 62 weeks versus 26.7% at 141 weeks). However, in LFD+CR mice, OTUs in the family Porphyromonadeceae (9.4% at 62 weeks versus 28.2% at 141 weeks), instead of bacteria in Bacteroides, were largely responsible for the increase of Bacteroidetes with age. Thus, the apparent phylum level changes associated with aging were actually mediated by different phylogenetic groups in animals with or without CR treatment.
We next used the Kendall tau rank correlation coefficient to directly measure the correlation between the phylotypes of gut microbiota in mid-life and the lifespan based on two types of diet. We identified that 45 OTUs significantly correlated with lifespan in mice fed on low-fat diet. Except for one Bacteroidales OTU, the remaining 15 OTUs significantly positively correlated with lifespan belonged to Firmicutes. Particularly, eight OTUs in Lactobacillus showed strong correlation with lifespan. The 30 phylotypes negatively correlated with lifespan were distributed in the five Phyla of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and TM7 ( and ). In the mice on the high-fat diet, 20 OTUs were positively correlated, and 18 OTUs were negatively correlated with lifespan, most of which were in Firmicutes and Bacteroidetes, except for two Actinobacteria OTUs ( and ). Because of the strong impact of different diet backgrounds on the gut microbiota, only three OTUs in Lactococcus (OTU65), Bacteroidales (OTU366) and Peptostreptococcaceae (OTU37) showed the same behaviour both in mice on low-fat diet and high-fat diet. These three OTUs were negatively correlated with lif...
The animal experimental procedures, approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, CAS, were described previously by Zhou et al. Male C57BL/6 J mice at 5 weeks of age were randomly assigned to one of the six groups ( n =30 for each group) and individually caged for a life-long trial: (1) low-fat diet with sedentary activity (LFD), (2) low-fat diet with 30% CR and sedentary activity (LFD+CR), (3) low-fat diet with voluntary running exercise (LFD+Ex), (4) high-fat diet with sedentary activity (HFD), (5) high-fat die with 30% CR and sedentary activity (HFD+CR) and (6) high-fat diet with voluntary running exercise (HFD+Ex). All faecal and serum samples for the current study were collected from this animal trial.
Machine-readable layer
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"text": "At the late-life age of 141 weeks, 27 OTUs were higher and 27 were lower in the LFD+CR group than in the LFD group ( and ). Ten of these OTUs were also significantly different between the two treatment groups in mid-life. For example, although OTU45 in the genus Lactobacillus was not the predominant phylotype in bacterial communities of LFD+CR mice, the relative abundance of this OTU was still higher in LFD+CR mice than in LFD mice (1.7% versus 0.024%, respectively; P <0.001, one-way ANOVA). Different from mid-life, the OTUs belonging to Bifidobacterium were higher in LFD+CR mice, but the OTU469 of Desulfovibrionaceae was lower in LFD+CR mice. Some members in the genus Bifidobacterium are well-known probiotic strains, and some in the family Desulfovibrionaceae have previously been found to be positively associated with obesity and inflammation."
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"text": "The mice had a significantly different gut microbiota structure between the LFD and HFD groups ( ), confirming results of previous studies. CR also shifted the gut microbiota in mice fed with high-fat diet but not as dramatic compared with their low-fat diet companions ( and ). In mid-life, 30 phylotypes were selected as key variables for separating the gut microbiota under different food intake conditions ( and ); 18 of them were higher and 12 were lower in the HFD+CR group than in the HFD group. All the phylotypes in Porphyromonadaceae were higher in the HFD+CR than in the HFD group. Most of the OTUs responding to CR in HFD+CR mice were not found in LFD+CR mice. Only three OTUs (in Lactococcus (OTU65), Bacteroidales (OTU366) and Peptostreptococcaceae (OTU37), respectively) were reduced, and three OTUs in Tannerella (OTU119, 155 and 267) were increased by CR both with mice fed with..."
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"text": "In LFD mice, the phylotypes mainly responsible for decrease of the phylum Firmicutes during aging were in the genus Allobaculum (12 OTUs; 28.1% at 62 weeks versus 0.20% at 141 weeks) ( and ). In LFD+CR mice, not only OTUs in the genus Allobaculum (29.7% at 62 weeks versus 7.2% at 141 weeks) but also OTUs in the genus Lactobacillus (21.0% at 62 weeks versus 2.0% at 141 weeks) made a significant contribution to the decrease of Firmicutes during aging ( and )."
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"name": "Wenjun Li"
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"name": "Shengyue Wang"
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