Results

Experimental molecular cell Biology

🧪 Results Summary: Brown Bear Serum & C. elegans

This chapter presents experimental results addressing two main questions:

Does brown bear serum differ by season (summer vs. winter) at the protein level?
Does brown bear serum affect muscle, mitochondrial health, motility, and stress responses in C. elegans?

The chapter progresses logically from proteomics → serum characterization → biological effects in worms.

🐻 6.1 Proteomic Analysis of Seasonal Differences in Brown Bear Serum

🎯 Purpose

To determine whether winter and summer brown bear serum differ in protein composition, particularly in ways relevant to hibernation physiology.

📊 6.1.1 About the Dataset

Previously published LC–MS/MS proteomics dataset
EDTA plasma from 14 free-ranging Swedish brown bears
Samples collected in both summer and winter (2014–2020)
113 samples total, measured in triplicate
Processed with MaxQuant, generating LFQ intensities

This dataset serves as background context for interpreting serum effects in C. elegans.

🧹 6.1.2 Data Processing

Performed in Perseus v2.1.5.0:

Initial identifications: 315 proteins
Removed:
- Contaminants
- Reverse hits
- Proteins identified only by site
Required presence in ≥70% of samples in at least one group
LFQ intensities:
- log₂-transformed
- Missing values imputed (width 0.3, downshift 1.8)

➡️ 110 proteins remained for downstream analysis

📈 6.1.3 Statistical Analysis

Two-way ANOVA
Two-sample t-tests
FDR correction = 0.05
Visualization:
- Volcano plot
- Heatmap

These analyses tested whether protein expression differs between seasons.

❄️🌞 6.1.4 Seasonal Differences Identified

68 proteins were significantly different between winter and summer
Heatmap showed consistent seasonal clustering
Confirms that bear serum proteome varies with season, consistent with previous hibernation studies

🧬 6.1.5 Proteins Relevant to C. elegans

Several seasonally regulated proteins have:
- Homologs, or
- Functional analogs in C. elegans

➡️ This supports the hypothesis that seasonal bear serum could cause measurable biological effects in worms

🧪 6.2 NanoDrop Analysis: Protein Concentration

📏 Total Protein Levels

Measured by NanoDrop A280:

Sample	Protein (mg/mL)
SBS	49.46
WBS	52.85
WBS blue	39.22

Winter serum ≈ 6.7% higher protein concentration
Matches trends seen in free-ranging bears

🌈 UV–Vis Spectra (220–750 nm)

280 nm peak → aromatic amino acids (Trp, Tyr, Phe)
220 nm peak → salts
WBS showed:
- ~2× higher 220 nm peak than SBS
- ~3× higher than WBS blue

➡️ Indicates distinct biochemical composition despite captivity

🧫 6.3 SDS-PAGE Protein Profiling

🔬 Method

SDS-PAGE + Coomassie Blue
Reducing conditions (DTT)
Dilutions: 1:10, 1:50, 1:100

🧾 Observations

Most bands similar across samples
Identified:
- Albumin (~66 kDa)
- IgG heavy chain (~50 kDa)
- IgG light chain (~25 kDa)

⚠️ WBS blue showed:

Strong bands at 15 kDa and 35 kDa
Likely hemoglobin monomers/dimers
Suggests partial hemolysis

🐛 6.4 Hatching Assay: Toxicity Screening

🥚 Question

Is bear serum toxic to C. elegans development?

✅ Result

90–100% survival
Worms reached young adult stage
Even undiluted serum was non-toxic

➡️ Confirms serum treatments are biologically tolerable

📊 6.5 Normality Testing

📐 Shapiro–Wilk Test

Applied to all datasets used in parametric statistics.

All assays:

p > 0.05
Normality not rejected

➡️ Parametric tests (ANOVA, t-tests) are valid

🏃 6.6 Motility: Thrashing Assay

🔍 Method

NL2099 worms
3- and 6-day adults
Automated body bend counts (30 s)

📊 Statistics

No Day × Serum interaction
Significant serum effect (p = 0.0012)
Data pooled across days

🚀 Key Result

Undiluted winter bear serum:
- +11% thrashing rate
- Significant vs PBS
All other treatments: not significant

➡️ Suggests enhanced muscle/mitochondrial function

🧱 6.7 Muscle Fiber Organization (Phalloidin Staining)

🎨 Method

Phalloidin staining of F-actin
Confocal microscopy
Z-stack projections
Muscle width measured (ventral & dorsal)

⚠️ Technical Issues

Freeze cracking caused:
- Broken worms
- Uneven staining

📊 Results

One-way ANOVA: global effect (p = 0.0288)
Dunnett’s post hoc vs PBS: no significant differences

➡️ At 3 days, muscle morphology changes are inconclusive ➡️ Age-related changes typically appear after ~7 days

🧬 6.8 β-Actin Expression (Western Blot)

🔬 Method

PD4251 worms
β-actin (42 kDa)
α-tubulin (55 kDa) loading control

📉 Findings

PBS control:
- Very high β-actin/α-tubulin ratio (~18)
Serum-treated worms:
- Much lower ratios (~2–3)

⚠️ One sample failed normalization ⚠️ Possible uneven stripping of α-tubulin

➡️ Suggests serum exposure may alter actin integrity, but needs replication

🔋 6.9 Mitochondrial Analysis (Myo-3::GFP)

🧠 Background

Mitochondrial shape reflects:
- Energy status
- Stress
- Fusion/fission balance

📐 6.9.1 Quantitative Morphology

Eccentricity (shape)

Lower eccentricity = more spherical
Pure SBS & WBS:
- Significantly lower eccentricity
Indicates fragmentation

Branch Density (networking)

All serum groups:
- Higher branch density than PBS
SBS 1:10 showed strongest effect

➡️ Paradoxical result:

Less tubular
More connected

👀 6.9.2 Qualitative Scoring (Blinded)

Categories:

Fragmented 🔴
Intermediate 🟢
Tubular 🔵

Results:

Pure serum → more fragmented
Diluted serum → more intermediate
Significant global effect (χ² p = 0.014)

➡️ Confirms quantitative analysis

🧪 6.9.3 Dauer Formation Assay

🧬 Rationale

Dauer entry reflects stress & metabolic state

🧫 Method

CB1370 strain
SDS resistance (1% SDS, 30 min)

📊 Results

All serum treatments reduced dauer formation
Highly significant:
- χ² p ≈ 6 × 10⁻⁸
- Odds ratio ≈ 0.32
Individual groups remained significant after Bonferroni correction

➡️ Indicates bear serum reduces stress signaling, regardless of season

🧠 Overall Takeaways

🐻 Bear serum differs by season at the proteomic level
🧪 Winter serum has higher protein content
🐛 Serum is non-toxic to C. elegans
🏃 Undiluted winter serum enhances motility
🧱 Muscle structure unchanged at early adulthood
🧬 Actin expression may be altered by serum
🔋 Mitochondria become more fragmented but more networked
😴 Serum reduces dauer formation, suggesting reduced stress

Quiz

Score: 0/31 (0%)

Q0. What was the primary purpose of the proteomic analysis of brown bear serum?

To identify novel proteins unique to captive bears

To compare seasonal differences in serum protein expression between summer and winter

To validate NanoDrop protein measurements

To assess toxicity of bear serum in C. elegans

Q1. Which technique was used to generate the proteomic dataset analyzed in the study?

Western blotting

ELISA

Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

2D gel electrophoresis

Q2. What filtering criterion was applied to retain proteins for downstream proteomic analysis?

Detected in 100% of all samples

Detected in at least 70% of samples in one seasonal group

Detected only in winter samples

Detected with p < 0.01 before correction

Q3. Why were LFQ intensities log2-transformed during data processing?

To increase the total number of proteins detected

To normalize the data distribution and stabilize variance

To eliminate missing values

To convert intensities to absolute protein concentrations

Q4. How many proteins were identified as significantly differentially expressed between summer and winter bears?

110

Q5. Which NanoDrop absorbance peak is primarily associated with aromatic amino acids?

220 nm

260 nm

280 nm

340 nm

Q6. What did the elevated 220 nm peak in winter bear serum primarily indicate?

Higher lipid concentration

Increased salt content

Protein degradation

Hemoglobin contamination

Q7. Which protein band at approximately 66 kDa observed in SDS-PAGE corresponds to a major serum protein?

IgG heavy chain

β-actin

Serum albumin

α-tubulin

Q8. What was the likely cause of the strong 15 kDa and 35 kDa bands in the WBS blue sample?

Increased actin expression

Partial hemolysis and free hemoglobin

Protease contamination

IgG degradation

Q9. What conclusion was drawn from the egg hatching assay in C. elegans?

Bear serum delayed development

Winter serum was toxic at high concentrations

Undiluted bear serum showed no significant toxicity

Only summer serum allowed normal hatching

Q10. Why was the Shapiro–Wilk test performed before applying ANOVA?

To test equality of variances

To test for independence of samples

To confirm normality of the data

To calculate effect size

Q11. Which treatment significantly increased thrashing rate in C. elegans?

Undiluted summer bear serum

Diluted winter bear serum

Undiluted winter bear serum

PBS control

Q12. Why were day-3 and day-6 thrashing data pooled?

Because both days showed identical thrashing rates

Because there was no significant Serum × Day interaction

To increase sample size for each group artificially

Because day-6 data failed normality testing

Q13. What limitation affected the interpretation of phalloidin muscle staining results?

Low antibody specificity

Freeze cracking caused uneven staining and damaged worms

Insufficient sample size

Incorrect microscope settings

Q14. Which result suggested that serum exposure may affect actin integrity despite unchanged muscle width?

Thrashing assay

Proteomic volcano plot

Western blot showing altered β-actin to α-tubulin ratios

NanoDrop protein concentration

Q15. The proteomic dataset used in the study was derived from captive brown bears.

True

False

Q16. Winter bear serum had a higher total protein concentration than summer bear serum.

True

False

Q17. The SDS-PAGE analysis was performed under reducing conditions using DTT.

True

False

Q18. Undiluted bear serum caused severe developmental defects in C. elegans embryos.

True

False

Q19. All datasets used for parametric testing failed the Shapiro–Wilk normality test.

True

False

Q20. Only diluted serum treatments increased thrashing rate in C. elegans.

True

False

Q21. Phalloidin staining showed clear, statistically significant increases in muscle width for serum-treated worms.

True

False

Q22. β-actin expression appeared lower in serum-treated worms compared to PBS controls after normalization.

True

False

Q23. Lower mitochondrial eccentricity values correspond to more spherical mitochondrial shapes.

True

False

Q24. All serum treatments increased mitochondrial branch density compared to PBS.

True

False

Q25. Diluted bear serum treatments were associated with more fragmented mitochondrial morphology.

True

False

Q26. Undiluted serum treatments tended to promote mitochondrial fragmentation.

True

False

Q27. Mitochondrial morphology in C. elegans is linked to stress and metabolic state.

True

False

Q28. Bear serum treatment increased the likelihood of dauer formation in C. elegans.

True

False

Q29. The SDS resistance assay identifies dauer larvae based on their survival in SDS.

True

False

Q30. The reduction in dauer formation suggests that bear serum may reduce stress signaling in C. elegans.

True

False