Nov 25, 2024
SENIOR Protocol
- J. Ignacio Serrano1,
- Silvia Cruz-Gil2,
- Cristina M. Fernández2,
- Lara P. Fernández2,
- Ana Sobrino1,
- Isabel Espinosa-Salinas2,
- Carolina Maestre3,
- María J. Latasa-Sada2,
- Manuel Cebrián1,
- Ricardo Ramos-Ruiz2,
- Ara Ramírez de Molina2,
- M. Dolores del Castillo1
- 1Spanish National Research Council (CSIC);
- 2IMDEA Food Institute;
- 3Centro Nacional de Investigaciones Oncológicas (CNIO)
Protocol Citation: J. Ignacio Serrano, Silvia Cruz-Gil, Cristina M. Fernández, Lara P. Fernández, Ana Sobrino, Isabel Espinosa-Salinas, Carolina Maestre, María J. Latasa-Sada, Manuel Cebrián, Ricardo Ramos-Ruiz, Ara Ramírez de Molina, M. Dolores del Castillo 2024. SENIOR Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9zmql5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 22, 2024
Last Modified: November 25, 2024
Protocol Integer ID: 112603
Abstract
Protocol to relate microbiota profiles with spontaneous brain activity in older people
Recruitment
Recruitment
The recruitment was performed through the GENYAL
Clinical Trials Platform at IMDEA Food Institute. This study was approved by
the institutional Research Ethics Committee (IMDEA Food Foundation) and
performed in accordance with the principles of research involving human
subjects stated in the Declaration of Helsinki (1964). All participants were
clearly informed about the study methodology and provided written informed
consent.
Inclusion criteria included: Age ≥ 55 years; BMI
between 27–35 kg/m2; able to understand the informed consent; willing to comply
with the study protocol. Exclusion criteria included: decreased cognitive function,
pregnancy or breastfeeding, severe chronic health conditions (heart, liver,
etc.), BMI >35 kg/m2, or pharmacological treatment for weight loss.
Anthropometric Measurements
Anthropometric Measurements
Anthropometric
measurements were collected following standardized methodology. Body
Composition Monitor analyzer (BF511- OMRON HEALTHCARE UK, LT, Kyoto, Japan) was
used to determine weight, % body fat, % skeletal muscle and visceral fat, as
well as basal metabolic rate.
Height was measured
with a stadiometer (Leicester-Biological Medical Technology SL, Barcelona).
Waist and hip
circumference (cm) were measured using Seca 201 (Quirumed, Valencia, Spain).
Blood pressure and pulse were monitored with the Model
M3 blood pressure monitor from OMRON HEALTHCARE UK, LT, Kyoto, Japan).
Behavioral Measurements
Behavioral Measurements
Sleep habits were assessed
via the Oviedo Sleep Questionnaire (OSQ). The OSQ is a
validated interview that support diagnosis of insomnia and hypersomnia during
the previous month based on the DSM-IV and ED-10 criteria. The questionnaire is
made up of a total of 15 items including Subjective satisfaction with sleep,
Insomnia (difficulty with falling or remaining asleep, early awakenings or
restorative sleep, among others), hypersomnia (daytime sleepiness and its
effects on daily tasks).
The psychological state of
the participants was measured using the short version of the Depression,
Anxiety and Stress Scale (DASS-21). designed to assess
the emotional states of depression, anxiety, and stress. The DASS-21 consists
of 21 items, divided into three subscales of 7 Likert items. A score for each
scale is calculated by summing all the related items and these scores are later
added to obtain a general score.
The Mini-Mental State
Examination (MMSE) (Spanish version) is a widely utilized cognitive screening
tool designed to assess cognitive impairment and dementia. The MMSE evaluate five cognitive domains:
orientation, registration, attention and calculation, recall, and language. The
total score has a maximum punctuation of 35, with lower scores indicating
greater cognitive impairment.
Fecal samples processing and 16S sequencing
Fecal samples processing and 16S sequencing
Fecal samples were sent to
Novogene for 16S sequencing of the bacterial V3V4 region: DNA was extracted by
using Magnetic Soil and Stool DNA Kit (TianGen, China, Catalog #: DP712),
following the manufacturer's protocol to ensure high-quality and high-yield DNA
suitable for downstream applications.
The 16S rRNA gene was
amplified using a set of universal primers targeting the V3-V4 regions of the
16S rRNA gene. The primers used were (5'- CCTAYGGGRBGCASCAG-3') and (5'-
GGACTACNNGGGTATCTAAT-3'), which are known for their broad coverage of bacterial
taxa while minimizing amplification of eukaryotic rRNA genes. The PCR
conditions were optimized to ensure specific amplification, with an initial
denaturation at 98°C for 1 minute, followed by 30 cycles of denaturation at
98°C for 10 seconds, annealing at 50°C for 30 seconds, and extension at 72°C
for 30 seconds, with a final extension at 72°C for 5 minutes.
The amplified 16S rRNA gene
fragments were performed by using specific primers connecting with barcodes.
The PCR products of proper size were selected through 2% agarose gel
electrophoresis. The same amount of PCR products from each sample was pooled,
end-repaired, A-tailed, and further ligated with Illumina adapters. Libraries
were sequenced on a paired-end Illumina platform to generate 250bp paired-end
raw reads.
Raw sequencing data were
processed using the DADA2 pipeline, which includes quality filtering,
dereplication, chimera removal, and sequence variant inference. This method
allows for the recovery of full-length 16S rRNA gene sequences with single-nucleotide
resolution and a near-zero error rate, ensuring high accuracy in microbial
community profiling.
Sequences were clustered
into Operational Taxonomic Units (OTUs) using a de novo clustering approach,
which has been shown to outperform reference-based methods in terms of
stability and quality of OTU assignments. The clustering was performed using
the VSEARCH algorithm, which is a viable open-source alternative to USEARCH.
Taxonomic classification of
the OTUs was performed using the SILVA database, which provides comprehensive
and up-to-date taxonomic information for 16S rRNA gene sequences. The
classification was done at various taxonomic levels, including phylum, class,
order, family, genus, and species.
EEG Data Acquisition
EEG Data Acquisition
The EEG signal was acquired
from 32 wet active ActiCAP electrodes (Brain Products GmbH, Gilching, Germany)
placed on the scalp in the positions Fp1, Fp2, AF3, AF4, F7, F3, Fz, F4, F8,
FC5, FC1, FC2, FC6, T7, C3, Cz, C4, T8, CP5, CP1, CP2, CP6, P7, P3, Pz, P4, P8,
PO3, PO4, O1, Oz, O2 according to the 10-20 system. Reference was set to the Fz
electrode and ground to the Fpz electrode. The signal was digitized and
recorded by a actiCHamp amplifier (Brain Products GmbH, Gilching, Germany) at
256Hz. The impedance of all electrodes was kept under 10 kΩ.
The participants were comfortably seated with their forearms resting in
their thighs, one meter away from a white wall.
They were asked to relax and breathe deeply with their mouth naturally
open:
3 minutes eyes closed
One minute free resting
3 minutes eyes closed
EEG Processing
EEG Processing
The EEG signal was preprocessed offline by a custom script in Matlab R2019b
(The MathWorks Inc., Natick, MA, USA) using the EEGLAB (Delorme & Makeig,
2004) functions
Load channel locations.
Resample to 256Hz.
ASR with burst rejection (std > 20).
Bandpass filter (1Hz-31Hz).
ICA.
ICLabel artifact rejection.
PREP pipeline for only bad channel detection and interpolation
Rereference to average (CAR).
Channel interpolation by spectrum std > 1.5
Source density in six frequency bands (theta: 4Hz-7Hz; low alpha:
7Hz-10Hz; high alpha: 10Hz-13Hz; low beta: 13Hz-18Hz; mid beta: 18.5Hz-21Hz;
high beta: 21Hz-30Hz) was calculated from the preprocessed signal by the
eLORETA algorithm implemented in the LORETA-KEY
software v20221229 .
Source density was averaged for each of the 210 cortical areas defined
by the Brainnetome atlas.