Amylin

Fibril antigens were prepared by stirring 2 mg/ml Aβ42 peptide in 50% HFIP/H2O, 0.02% sodium azide for 7 days. Afterwards, the HFIP was evaporated under a stream of nitrogen and the sample was stirred for an additional 7 days and dialyzed against PBS (molecular weight cut off 10,000 Da). The resulting fibrils were checked by EM and the purity was confirmed by the absence of oligomers using anti-oligomer antibody. The same protocol was used to prepare IAPP fibrils. Aβ and IAPP oligomer mimics were prepared as previously described [27 (link)] The synthesis of IAPP C-terminal thioester analog lacking cysteine residues (KANTATAATQRLANFLVHSSNNFGAILSSTNVGSNTY-SR), was also carried out according to the methods described [27 (link)]. The antigens were each used to immunize two New Zealand white rabbits (Pacific Immunology Corp., Ramona, CA, 92065) according to protocols approved by IACUC. Each rabbit immunized with 500 μl of antigen in complete Freund's adjuvant (CFA), and then boosted twice at four week intervals with 500 μl of antigen in Incomplete Freund's Adjuvant (IFA).

Our model of FS cells is based on that of [3 (link),28 (link)], with several modifications based on voltage clamp data. The current balance equation is
where V is the membrane potential of the neuron, C = 1μF/cm² is the membrane capacitance, and the parameters of the leak current are g_L = 0.25 mS/cm² and V_L = −70 mV. The external current injected into the neuron is denoted by I_app.
The Na⁺ current I_Na is given by:
where the gating variables, h and m, follow:



The parameters are: g_Na = 112.5 mS/cm², V_Na = 50 mV, σ_m = 11.5 mV, θ_h = −58.3 mV, σ_h = −6.7 mV, θ_th = −60 mV, σ_th = −12 mV [14 (link)]. In this work, we study the effect of the strength of the Na⁺ window current, controlled by the parameter θ_m, on the dynamics of the neuron.
The delayed rectifier K⁺ current I_Kdr is of the Kv3.1–Kv3.2 type. It is responsible for the brief duration of the spike, about 0.5 ms [2 (link),48 (link)], and for the high firing frequency [3 (link),49 (link)]. It is given by:
with:


All the parameters of the delayed rectifier current are fixed: g_Kdr = 225 mS/cm², V_K = −90 mV, θ_n = −12.4 mV, σ_n = 6.8 mV, θ_tn = −27 mV, σ_th = −15 mV [50 (link)].
The K⁺ current I_d incorporated in the model [10 (link),11 (link)] has fast activation and slow inactivation. It is defined by:




Throughout the paper, all the parameters of the d-current but g_d are fixed: θ_a = −50 mV, σ_a = 20 mV, τ_a = 2 ms, θ_b = −70 mV, σ_b = −6 mV, τ_b = 150 ms [51 (link),52 (link)]. The parameter g_d is varied to study the effect of the strength of this current.
Finally, to study the effect of noise in the external input on the firing pattern of the neuron, we add an additional external input, I_noise, of the form:
where ξ(t) is a Gaussian white noise with an average 0 and a unit variance, and D has the units of μA² × ms/cm⁴ .
Numerical methods. Simulations were performed using the fourth-order Runge-Kutta method with a time step of 0.01 ms implemented as a C program or within the software package XPPAUT [53 ], which was used also for computing bifurcation diagrams.
Delay. The delay duration t_delay is defined to be the time from the onset of current injection, or, if the neuron fires transient 1–3 spikes, from the last transient spike to the first spike of the sustained firing. We define that the neuron shows a delay if t_delay is at least twice as large as the inter-spike interval during steady-state spiking t_ISI, or if it is larger than both 100 ms and 1.2 t_ISI.
Fourier spectrum. Discrete Fourier transforms of subthreshold oscillations were calculated numerically over a time window of T_FT ending T_BS = 5 ms before the first spike of the steady-state firing. The absolute values of the Fourier components were averaged over n_R repetitions of the same stimulus. Parameters for Figure 9 are: T_FT = 120 ms, n_R = 20. Parameters for Figure 12 are: T_FT = 90 ms, n_R = 5 (A), T_FT = 120 ms, n_R = 13 (B), T_FT = 120 ms, n_R = 11 (C).

Cell Ranger (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) and Seurat (https://satijalab.org/seurat/) were applied to cluster and identify the cell types based on known marker expression. The marker genes were PRSS1, CTRB1, CTRB2, and REG1B for acinar cells, MS4A1, CD79A, CD79B, and CD52 for B cells, AMBP, CFTR, and MMP7 for type1 ductal cells, KRT19, KRT7, TSPAN8, and SLPI for type 2 ductal cells, CHGB, CHGA, INS, and IAPP for endocrine cells, CDH5, PLVAP, VWF, and CLDN5 for endothelial cells, LUM, DCN, and COL1A1 for fibroblast cells, AIF1, CD64, CD14, and CD68 for macrophage cells, ACTA2, PDGFRB, and ADIRF for stellate cells, and CD3E, CD4, and CD8 T cells (21 (link)). The reads for each cell type were extracted and combined. RNA editing events for each type were identified by REDItools. Then, credible RNA editing sites were filtered by satisfying stringent requirements (total reads for each site in each sample ≥10, 1 > editing level for each site in each sample ≥0.1, remove SNP sites).

This study comprised 100 adult patients with T2D and 50 age- and sex-matched healthy controls. Patients with T2D were consecutively recruited from Diabetes Outpatient Clinic at Mansoura Specialized Medical Hospital, Mansoura University, Mansoura, Egypt. The inclusion criteria were patients with duration of T2D > 10 years and who had symptoms of gastroparesis [Gastroparesis Cardinal Symptom Index (GCSI) Score ≥ 1.9]. Patients with T2D were submitted for transabdominal ultrasonography accordingly, they divided into 2 groups: patients with gastroparesis (n = 55) and patients without gastroparesis (n = 45). Exclusion criteria were history of digestive tract surgery or prior gastric outlet obstruction, thyroid disease, liver & renal failure, neuropsychiatric disorder, connective tissue disorders, malignancies, pregnancy and participants taking vitamin B12 and alcohol. Drugs that could potentially interfere with gastrointestinal motility such as GLP-1 receptor agonists and the amylin analog, α glucosidase inhibitors, and opioid analgesic were also excluded. Healthy controls were recruited from the same geographic area with the same exclusion criteria.
All participants were subjected to a thorough medical history and underwent a clinical examination. Anthropometric measurements including height, body weight, body mass index (BMI) (kg/m²), and waist circumference (WC) were obtained using standardized techniques. The diagnosis of diabetic gastroparesis was based on the symptom validated questionnaire GCSI Score ≥ 1.9 and ultrasonographic findings including gastric emptying ˂ 35.67% and motility index ˂ 5.1. The cut-off point of gastric emptying and motility index were calculated from our study healthy controls as mean—2SD.
The GCSI Score consists of 9 symptoms covering 3 areas; nausea/vomiting subscale (3 symptoms: nausea, vomiting and retching), postprandial fullness/early satiety subscale (4 symptoms: stomach fullness, early satiety, postprandial fullness and loss of appetite) and bloating subscale (2 symptoms: bloating and stomach distension). All symptoms are rated from 0 to 5 over the prior 2 weeks [no symptoms = 0, very mild = 1, mild = 2, moderate = 3, severe = 4, and very severe = 5]. GCSI Score was calculated as the average of the 3 symptom subscales [17 (link)]. The clinical severity of gastroparesis was graded on a scale originally proposed by Abell et al. [18 (link)]; grade 1: mild gastroparesis (symptoms are relatively easily controlled and weight and nutrition can be maintained with a regular diet); grade 2: compensated gastroparesis (symptoms are partially controlled with the use of daily medications and nutrition can be maintained with dietary adjustments); grade 3: gastroparesis with gastric failure (uncountable refractory symptoms with frequent hospitalizations and/or inability to maintain nutrition via an oral route).
Vitamin B12 deficiency was defined as vitamin B12 levels below 125 pmol/L [16 (link)]. Peripheral neuropathy was diagnosed based on neuropathy disability and symptom scores [19 (link), 20 (link)]. Diabetic nephropathy was diagnosed according to Umanath & Lewis [21 (link)]. Diabetic retinopathy was assessed through fundus examination.

Mouse islets were isolated from heterozygous human islet amyloid polypeptide (hIAPP) transgenic (hIAPP^+/-) 8-12-week-old FVB/N mice (Jackson Laboratory, Bar Harbor, ME, USA). The Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute approved all animal experimental protocols in this study. Human umbilical cord blood- (hUCB-) MSCs were isolated according to a reported method [23 (link)]. Umbilical cord blood (UCB) units obtained from full-term deliveries were collected from the unborn placenta with the informed consent of the mothers. The human UCB-MSC isolation procedure was approved by the Institutional Review Board of Samsung Medical Center (IRB No. SMC 2019-11-026), and all participants provided informed consent for the use of the umbilical cord in this experimental study. The human monocyte cell line THP-1 was purchased from the Korean Cell Bank (Seoul, Korea) and maintained in complete RPMI 1640 media.