Meridians

Aggregate retinotopic maps of each dataset were produced separately for polar angle and eccentricity by finding the weighted mean polar angle and eccentricity of all subjects at each aligned vertex position. Mean polar angles and eccentricities were weighted by the F-statistic of the confidence of each subject's polar angle and eccentricity assignments. A confidence for each vertex in the aggregate was calculated as the sum of squares of the F-statistics of all significant vertices divided by the sum of the same F-statistics. For a set of subjects Q, each of whom have a vertex at position p on the cortical surface with a polar angle and eccentricity assignment whose significance is above threshold, the confidence of aggregate vertex p is (Σ_q•QF(q, p)²)/(Σ_q•QF(q, p)) where F(s, x) is the confidence of the polar angle and eccentricity assignment in subject s at vertex position x. The assignment of any vertex whose confidence was below a minimum threshold chosen for the dataset (see Supplemental Mathematica Notebook, §3.2), was discarded. Because averaging produces bias in the direction of the mean near the borders of a finite stimulus range (e.g., values near 0° and 180° of polar angle tend to attenuate toward 90° in the aggregate), the aggregate polar angle values were corrected and eccentricity was truncated by 1.25°. Polar angle correction was performed by forcing the distribution of polar angles in the corrected aggregate to match the distribution of the union of all significant polar angle values of all subjects. More specifically, the uncorrected aggregate polar angle θ of each vertex in the aggregate was changed to a corrected polar angle θ′ such that C(A, θ) = C(M, θ′) where C(D, t) is the cumulative density function of the distribution D, evaluated at t, and A and M are the distributions of the uncorrected aggregate polar angles and union of all significant polar angle values for all subjects, respectively. Eccentricity values below 1.25° and within 1.25° of the outer stimulus border were excluded due to measurement bias near the edge of the stimulus range [23] (link).
All vertices within π/3 radians on the inflated spherical hemisphere of the point p₀, defined as the most anterior point on the anatomically defined V1 border [7] (link), were rotated such that p₀ lay at the intersection of the equator of the spherical fsaverage_sym brain hemisphere and prime meridian, then flattened via projection onto the plane tangent to the sphere at p₀. A shear transformation, present also in our previous treatment of V1 [11] (link), was applied to the flattened data to render the V1 region more elliptical. These flattened and sheared data formed a “flattened occipital region” on the cortical surface.

Frozen liver tissues were homogenised in 400 µl of ice-cold RIPA buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA pH 8.0, 1 mM NaF, 0.1% SDS, 1% Triton X-100, 1% Sodium Deoxycholate with freshly added 1 mM NaVO₄ and protease inhibitors) using a PowerGen 125 homogeniser and lysates normalised to 1 µg per 1 µl. Proteins were separated on a 4–12% Bis–Tris gel by SDS-PAGE and transferred onto nitrocellulose membrane.
Membranes were probed for the following; phospho-AKT (Ser 473, cat: 4060), total Akt (cat: 4691), phospho-S6 (Ser 235/236, cat: 4858), total S6 (cat: 2217), phospho-AMPK (Thr 172, cat: 2535), total AMPK (cat: 5832) and GAPDH (cat: 5174) (all Cell Signaling Technology), DEGS1 (cat: ab185237, Abcam), RBP4 (Dako), or IR β-chain (Santa Cruz Biotechnology). ApoB 48, ApoB 100 (Meridian Life Sciences UK, cat: K23300R) and Vinculin (Cell Signaling Technology, cat: 13901) were separated on a 6% Tris–Glycine gel. Anti-rabbit and anti-mouse horse radish peroxidase (HRP) conjugated antibodies were from Anaspec. Primary and secondary antibodies were used at 1:1000 and 1:5000 respectively.
Blots used in figures are all compliant with the digital image and integrity policies of Nature publishing and Scientific Reports journal. Western blot membranes were cut at approximate molecular weight (± 20 kDa) of target protein before incubation of primary antibodies. Equal numbers of representative samples from all treatment groups were run on multiple gels/blots to accommodate all samples. Images obtained were minimally processed. Image analysis and quantification with normalisation to loading control protein was performed within the same membrane and then data combined for graphical representation. No direct quantitative comparisons between samples on different gels/blots were performed.

The study will take place in the Outpatient Department, Shanghai Research Institute of Acupuncture and Meridian, Shanghai, China, and the recruitment will be announced via our official account on social media. Placement of brochures and study posters in Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine and Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (collaborating hospitals), and community centers will be used to assist recruitment. Participants or their representatives will be instructed to read the informed consent and those who showed interest in the study will be scheduled for the screening visit. After presenting written informed consent, participants will be examined, and if all entry criteria are met, will commence a 4-week run-in period of treatment with donepezil hydrochloride (5 mg/tablet, China) 5 mg daily (prior AD treatments will be terminated by then). To further avoid potential confounding effects on the clinical outcomes and gut microbiota, participants will be instructed to maintain their regular diet and activity level, and refrain from probiotics, prebiotics, or synbiotics use. Upon successful completion of the 4-week run-in, participants will be scheduled to complete baseline assessments and undergo randomization. The study flowchart and proposed trial schedule are shown in Figure 1 and Table 1, respectively.