🐭 实验小鼠组别详解（中文版）
以下是本研究中使用的全部 14 个实验组别的详细说明，按功能分类整理：
🔹 第一类：中风模型组（用于图 4 和补充图 3）
| 组号 | 样本前缀 | 完整样本 | 性别/年龄 | 状态 | 用途 |
|---|---|---|---|---|---|
| 1 | sample-A* |
A1–A11 | ♂ 老年 | 中风后 3 天 | 图 4D–F（血液/脑组织 SCFA 检测） |
| 2 | sample-B* |
B1–B16 | ♀ 老年 | 中风后 3 天 | 图 4D–F（血液/脑组织 SCFA 检测） |
📌 说明：这两组用于比较中风后老年雄性和雌性小鼠的微生物代谢物（短链脂肪酸）水平差异。
🔹 第二类：基线供体组（用于图 4、补充图 4、图 5C）
| 组号 | 样本前缀 | 完整样本 | 性别/年龄 | 状态 | 用途 |
|---|---|---|---|---|---|
| 3 | sample-C* |
C1–C10 | ♀ 老年 | 基线，FMT 供体 | 图 4A–C（16S 测序）、补充图 4、图 5C（Boxplot 3） |
| 4 | sample-E* |
E1–E10 | ♂ 老年 | 基线，FMT 供体 | 图 4A–C（16S 测序）、补充图 4、图 5C（Boxplot 2） |
| 5 | sample-F* |
F1–F5 | ♂ 年轻 | 基线，FMT 供体 | 对照供体，未在主图中展示 |
📌 关键说明：
- 组 3 和组 4 是粪菌移植（FMT）的供体小鼠，用于提供老年雌/雄肠道菌群
- 图 4 和补充图 4 中实际使用的样本为：♀供体 C1–C6（n=6），♂供体 E1–E8（n=8），其余样本因年龄偏小或测序深度不足被排除
🔹 第三类：FMT 预处理组（用于图 5B 紫色点）
| 组号 | 样本前缀 | 完整样本 | 性别/年龄 | 状态 | 用途 |
|---|---|---|---|---|---|
| 6 | sample-G* |
G1–G6 | ♂ 老年 | FMT 前，抗生素处理前，批次 I | 图 5B（紫色，pre-FMT 基线） |
| 7 | sample-H* |
H1–H6 | ♀ 老年 | FMT 前，抗生素处理前，批次 I | 图 5B（紫色，pre-FMT 基线） |
| 8 | sample-I* |
I1–I6 | ♂ 年轻 | FMT 前，抗生素处理前，批次 II | 图 5B（紫色，pre-FMT 基线） |
📌 说明：这三组合并为”pre-FMT”基线组（n=18），代表年轻雄性受体小鼠在接受粪菌移植之前的肠道菌群状态。
🔹 第四类：FMT 受体组（用于图 5）
| 组号 | 样本前缀 | 完整样本 | 性别/年龄 | 接受供体 | 状态 | 用途 |
|---|---|---|---|---|---|---|
| 9 | sample-J* |
J1–J4, J10, J11 | ♂ 年轻 | 老年♂供体 | FMT 后，中风前 | 图 5B🔵、5C（Boxplot 4）、5D、5E |
| 10 | sample-K* |
K1–K6 | ♂ 年轻 | 老年♀供体 | FMT 后，中风前 | 图 5B🔴、5C（Boxplot 5）、5D、5E |
| 11 | sample-L* |
L2–L6 | ♂ 年轻 | 年轻♂供体 | FMT 后，中风前 | 图 5B🟢、5E（对照） |
📌 关键说明：
- 所有受体均为年轻雄性小鼠，仅供体来源不同
- “aged♂ FMT” = 接受老年雄性供体粪便的年轻受体（不是受体本身是老年！）
- 图 5C 的 5 个箱线图 = pre-FMT 基线 + 2 个供体组 + 2 个受体组（不含年轻♂供体受体组）
🔹 第五类：FMT + 中风后组（未在主图展示）
| 组号 | 样本前缀 | 完整样本 | 性别/年龄 | 接受供体 | 状态 | 用途 |
|---|---|---|---|---|---|---|
| 12 | sample-M* |
M1–M8 | ♂ 老年 | 老年♂供体 | FMT 后，中风后 | 补充分析 |
| 13 | sample-N* |
N1–N10 | ♀ 老年 | 老年♀供体 | FMT 后，中风后 | 补充分析 |
| 14 | sample-O* |
O1–O8 | ♂ 年轻 | 年轻♂供体 | FMT 后，中风后 | 补充分析 |
📌 说明：这三组用于探索性分析，未出现在主论文图表中。
🧭 快速记忆口诀
✅ "FMT 标签 = 供体特征，不是受体特征"
• aged♂ FMT = 供体是老年雄性
• 受体永远是年轻雄性（本实验设计）
✅ 图 4 = 老年供体（组 3/4）+ 老年中风小鼠（组 1/2）
✅ 图 5 = FMT 实验：受体（组 6–11）+ 供体（组 3/4）
✅ 补充图 4 = 仅老年供体（组 3/4，筛选后 C1–C6, E1–E8）⚠️ 样本排除说明
| 组别 | 排除样本 | 排除原因 |
|---|---|---|
| 组 3（♀供体） | C7, C8, C9, C10 | C8–C9 年龄偏小；C10 为离群值/测序深度低 |
| 组 4（♂供体） | E9, E10 | 测序深度低/离群值 |
| 组 9（受体） | J5, J6, J7, J8, J9 | 测序深度不足或质量控制排除 |
| 组 10（受体） | K7–K15 | 测序深度不足或质量控制排除 |
| 组 11（受体） | L1, L7–L15 | 测序深度不足或质量控制排除 |
📌 最终用于分析的样本数以各图图例标注为准（如：图 5 中 aged♂ FMT n=6, aged♀ FMT n=6）
TODO: 导出完整的样本–组别映射 CSV 文件，or 提供某张图的精确样本列表🎯
关于 “aged♂ FMT” 的明确解释
aged♂ FMT = 接受了老年雄性供体粪便的年轻雄性受体小鼠
🔹 实验设计核心逻辑
| 角色 | 年龄/性别 | 说明 |
|---|---|---|
| 受体（接受粪便） | 🐭 年轻雄性（4周龄起始） | 所有 FMT 组的受体都是相同的年轻雄性小鼠 |
| 供体（提供粪便） | 🐭 老年雄性 / 老年雌性 / 年轻雄性 | 供体的年龄/性别是实验变量 |
🔹 样本分组详解
🟣 Purple (pre-FMT, n=18): G1–G6, H1–H6, I1–I6
→ FMT前的基线年轻雄性小鼠（未接受移植）
🔵 Blue (aged♂ FMT, n=6): J1, J2, J3, J4, J10, J11
→ 年轻雄性受体 + 接受【老年雄性】供体粪便
🔴 Red (aged♀ FMT, n=6): K1–K6
→ 年轻雄性受体 + 接受【老年雌性】供体粪便
🟢 Green (young♂ FMT, n=5): L2–L6
→ 年轻雄性受体 + 接受【年轻雄性】供体粪便（对照组）🔹 文献依据
来自 260311_LTPaper.pdf Figure 5 图例：
“Principal coordinates analysis (PCoA) of young male mice before (purple) (n=18), and after FMT of aged male (n=6) (blue) or female (n=6) (red) or young male (n=5) (green) stool donors.”
→ 明确说明分析对象是 young male mice，括号内描述的是 stool donors（粪便供体）的特征。
来自 Supplemental Methods “Microbiota eradication and FMT”：
“4 weeks old male mice were treated for 2 weeks with an antibiotic cocktail… recipient mice were gavaged with donor stool four times over two weeks.”
→ 受体小鼠起始年龄为 4周龄（年轻）。
Figure 5 小标题：
“FMT of aged male microbiota increases IL-17A-producing γδ T cells in the post-ischemic brain of young recipient mice“
→ 再次确认受体是 young recipient mice。
🔹 为什么这样设计？
这个实验的核心科学问题是：
“供体微生物的年龄/性别特征，能否通过移植’传递’给受体，并影响受体的免疫反应？”
通过保持受体一致（年轻雄性），仅改变供体来源，可以：
- 排除受体自身年龄/性别的混杂效应
- 直接评估供体微生物对受体免疫表型（如 IL-17A⁺ γδ T 细胞）的因果影响
- 验证”微生物介导的年龄/性别差异”假说
✅ 快速记忆口诀
“FMT 标签 = 供体特征，不是受体特征”
- aged♂ FMT = 供体是老年雄性
- 受体永远是年轻雄性（本实验中）
🔹 Figure 5B: PCoA of FMT Experiment
“Principal coordinates analysis (PCoA) of young male mice before (purple) (n=18), and after FMT of aged male (n=6) (blue) or female (n=6) (red) or young male (n=5) (green) stool donors.”
- 🟣 Purple (pre-FMT, n=18): Groups 6+7+8 →
G1–G6,H1–H6,I1–I6 - 🔵 Blue (aged♂ FMT, n=6): Group9 →
J1,J2,J3,J4,J10,J11 - 🔴 Red (aged♀ FMT, n=6): Group10 →
K1–K6 - 🟢 Green (young♂ FMT, n=5): Group11 →
L2–L6(L1, L7–L15 excluded for low depth/QC)
🔹 Figure 5C=Figure 5B+C1-7+E1-10 (Need to be confirmed?): Family-Level Relative Abundance Boxplots (5 panels)
Based on your co-author’s note: “Figure 5C uses the Figure 5B recipient samples PLUS the aged donor samples (Groups 3 & 4).”
- Boxplot 1 (pre-FMT baseline, n=18): Groups 6+7+8 →
G1–G6,H1–H6,I1–I6 - Boxplot 2 (aged♂ stool donors, n=8): Group4 →
E1–E10 - Boxplot 3 (aged♀ stool donors, n=6): Group3 →
C1–C7 - Boxplot 4 (aged♂ FMT recipients, n=6): Group9 →
J1,J2,J3,J4,J10,J11 - Boxplot 5 (aged♀ FMT recipients, n=6): Group10 →
K1–K6 - !!No Group11 (L2-L6)!!
⚠️ Key difference: Group11 (young♂ FMT recipients,
L2–L6) is shown in Figure 5B but is NOT included in Figure 5C, since Figure 5C focuses on comparing the effect of aged donor microbiota.
🔹 Figure 5D: Bubble Plot of Differentially Abundant Taxa (DESeq2)
“Bubble plot showing differentially abundant Operational Taxonomic Units (OTUs) between young male recipients of aged female vs. aged male FMT. x-axis = log₂ fold change, y-axis = bacterial family, bubble size = adjusted p-value, color = bacterial order.”
- 🔵 Aged♂ FMT recipients (Group9, n=6):
J1,J2,J3,J4,J10,J11→ Reference group (log₂FC < 0 = enriched in this group) - 🔴 Aged♀ FMT recipients (Group10, n=6):
K1–K6→ Comparison group (log₂FC > 0 = enriched in this group)
| Key families highlighted in the plot: | Direction | Family (Order) | Enriched in | Biological note |
|---|---|---|---|---|
| 🔴 Positive log₂FC | Lachnospiraceae (Clostridiales) | Aged♀ FMT | SCFA producer | |
| 🔴 Positive log₂FC | Ruminococcaceae (Clostridiales) | Aged♀ FMT | SCFA producer | |
| 🔴 Positive log₂FC | Muribaculaceae (Bacteroidales) | Aged♀ FMT | SCFA producer | |
| 🔴 Positive log₂FC | Desulfovibrionaceae (Desulfovibrionales) | Aged♀ FMT | Sulfate-reducing | |
| 🔵 Negative log₂FC | Erysipelotrichaceae (Erysipelotrichales) | Aged♂ FMT | Pro-inflammatory association | |
| 🔵 Negative log₂FC | Rikenellaceae (Bacteroidales) | Aged♂ FMT | Context-dependent | |
| 🔵 Negative log₂FC | Clostridiales vadinBB60 group | Aged♂ FMT | Function unclear |
⚠️ Note: This analysis uses DESeq2 on non-rarefied integer counts from
ps_filt, with taxa prefiltered (total counts ≥10). Only taxa with Benjamini–Hochberg adjusted p < 0.05 are shown. The same ASVs/OTUs appear in Figure 4C and Supplementary Figure 4B, but Figure 5D specifically compares FMT recipient outcomes (Groups 9 vs. 10), not baseline donor differences.
🔹 Supplementary_Figure4=Figure4B-C: Aged Donors (Homeostatic)
“(A) Bray-Curtis distances between aged male-male, female-female and female-male stool samples under homeostatic conditions (nmale=8 and nfemale=6). (B) Cladogram showing differentially abundant OTUs…”
- 👨 Aged male donors (n=8): Group4 →
E1–E8(E9, E10 excluded for low sequencing depth/outliers) - 👩 Aged female donors (n=6): Group3 →
C1–C6(C7–C10 excluded; C8–C9 younger mice, C10 outlier)
🔹 Figure 4B-C: Sex Differences in Aged Mice (16S rRNA-seq panels B–C)
“We profiled the gut bacterial composition of aged male and female mice by 16S rRNA-seq…”
- Baseline aged female donors: Group3 →
C1–C6 - Baseline aged male donors: Group4 →
E1–E8
(Note: Figure 4D–F show SCFA concentrations measured by targeted UHPLC-MS/MS, not 16S data.)
✅ PICRUSt2 NOT used in Figure 4D–F
Your observation is CORRECT: PICRUSt2 results are NOT used in Figure 4D–F.
| Question | Answer | Evidence |
|---|---|---|
| Are PICRUSt2 results used in Figure 4? | ❌ No | Figure 4D–F legend explicitly states: “measured by targeted mass spectrometry” |
| Are PICRUSt2 results used anywhere in the manuscript? | ❌ No evidence | README_PICRUSt2.txt files contain exploratory pipeline notes, but no PICRUSt2 figures, tables, or text appear in 260311_LTPaper.pdf or 260310_Supplements.pdf |
| Is the SCFA data in Figure 4D–F experimentally measured? | ✅ Yes | Supplemental Methods (pages 12–13) describe UHPLC-MS/MS quantification with internal standards, derivatization, and MRM parameters |
Key distinction:
- PICRUSt2 → Predicts functional potential (gene/pathway abundances) from 16S sequences; outputs are relative, unitless values.
- Figure 4D–F → Measures actual SCFA concentrations (acetate, butyrate, etc.) in µmol/l via targeted mass spectrometry; outputs are absolute, quantitative values.
Here is the merged quick reference table combining Figure 5B, 5C, and 5D with related figures, formatted for easy copy-paste:
🔹 Quick Reference: All Figure 5 Panels vs. Related Figures
| Figure | Comparison | Sample IDs (exact) | n | Purpose |
|---|---|---|---|---|
| Figure 4B-C | Aged♀ vs. aged♂ donors (homeostatic) | C1–C6 vs. E1–E8 |
6 vs. 8 | Baseline sex differences in microbiota (DESeq2 bubble plot) |
| Suppl Fig 4B | Same as Fig 4C | C1–C6 vs. E1–E8 |
6 vs. 8 | Phylogenetic context of differential taxa (cladogram) |
| Figure 5B | Pre-FMT vs. post-FMT recipients (4-group PCoA) | G1–G6, H1–H6, I1–I6 (pre-FMT); J1, J2, J3, J4, J10, J11 (aged♂ FMT); K1–K6 (aged♀ FMT); L2–L6 (young♂ FMT) |
18, 6, 6, 5 | PCoA: microbiome shift after FMT (Bray–Curtis) |
| Figure 5C | Donors vs. recipients (5 boxplots, family-level) | G1–G6, H1–H6, I1–I6 (pre-FMT); E1–E8 (aged♂ donors); C1–C6 (aged♀ donors); J1, J2, J3, J4, J10, J11 (aged♂ FMT); K1–K6 (aged♀ FMT) |
18, 8, 6, 6, 6 | Taxonomic composition: donors vs. recipients (relative abundance) |
| Figure 5D | Aged♀ vs. aged♂ FMT recipients (DESeq2) | K1–K6 vs. J1, J2, J3, J4, J10, J11 |
6 vs. 6 | Effect of donor microbiota on recipient immune response (differential abundance) |
| Figure 5E | Same recipients as Fig 5D (+ young♂ control) | K1–K6 vs. J1, J2, J3, J4, J10, J11 (+ L2–L6) |
6 vs. 6 (+5) | IL-17A+ γδ T cells in brain post-FMT (flow cytometry) |
🔹 Sample-ID Master List for Figure 5
| Group # | Description | Sample Prefix | Full IDs | Used In |
|---|---|---|---|---|
| 3 | Aged female, baseline FMT donor | sample-C* |
C1–C10 (C1–C6 used in Fig 4B-C, Suppl Fig 4, Fig 5C) | Fig 4C, Suppl Fig 4, Fig 5C |
| 4 | Aged male, baseline FMT donor | sample-E* |
E1–E10 (E1–E8 used in Fig 4B-C, Suppl Fig 4, Fig 5C) | Fig 4B-C, Suppl Fig 4, Fig 5C |
| 6 | Aged male, pre-antibiotics FMT batch I | sample-G* |
G1–G6 | Fig 5B (purple), Fig 5C (Boxplot 1) |
| 7 | Aged female, pre-antibiotics FMT batch I | sample-H* |
H1–H6 | Fig 5B (purple), Fig 5C (Boxplot 1) |
| 8 | Young male, pre-antibiotics FMT batch II | sample-I* |
I1–I6 | Fig 5B (purple), Fig 5C (Boxplot 1) |
| 9 | Young male, post-FMT aged male stool | sample-J* |
J1–J4, J10, J11 (J5–J9 excluded) | Fig 5B (blue), Fig 5C (Boxplot 4), Fig 5D, Fig 5E |
| 10 | Young male, post-FMT aged female stool | sample-K* |
K1–K6 | Fig 5B (red), Fig 5C (Boxplot 5), Fig 5D, Fig 5E |
| 11 | Young male, post-FMT young male stool | sample-L* |
L2–L6 (L1, L7–L15 excluded) | Fig 5B (green), Fig 5E (not in Fig 5C/D) |
🔹 Key Notes for Interpretation
- Figure 5B vs. 5C: Figure 5B shows beta-diversity (PCoA) of all FMT groups; Figure 5C shows taxonomic composition (boxplots) of donors + recipients. Group11 (young♂ FMT) is in 5B but not in 5C.
- Figure 5D: Uses DESeq2 on non-rarefied counts from
ps_filt(taxa prefiltered: total counts ≥10). Only taxa with BH-adjusted p < 0.05 are shown. - Figure 5E: Includes the same recipients as Fig 5D plus the young♂ FMT control group (Group11,
L2–L6) for comparison of IL-17A+ γδ T cells. - Sample exclusions: C7–C10, E9–E10, J5–J9, K7–K15, L1, L7–L15 were excluded for low depth, outliers, or QC reasons (see README files).
Let me know if you’d like me to:
- Export the exact DESeq2 results table for Figure 5D as CSV/Excel,
- Provide the R code snippet that generates the bubble plot for Figure 5D, or
- Draft the full email reply to your colleague with these merged tables integrated. 🎯