κ‑Casein-Based Hierarchical Suprastructures and Their Use for Selective Temporal and Spatial Control over Neuronal Differentiation

Functions are diversified by producing hierarchical structures from a single raw material. Biologically compatible milk protein of κ-casein has been employed to fabricate higher-order suprastructures. In the presence of dithiothreitol and heat treatment, κ-casein transforms into amyloid fibrils with distinctive morphology attributable to mechanism-based fibrillar polymorphism. As the fibrils elongate to yield high aspect ratio during high-temperature incubation, the resulting fibrils laterally associate into the liquid crystalline state by forming a two-dimensional fibrillar array. Following a desalting process, the fibrillar arrays turn into a three-dimensional matrix of hydrogel that could be selectively disintegrated by subsequent salt treatment. The hydrogel was demonstrated to be a matrix capable of exhibiting controlled release of bioactive substances like retinoic acid, which led to temporal and spatial control over the differentiation of neuronal cells. Therefore, the hierarchical suprastructure formation derived from the single protein of κ-casein producing one-dimensional protein nanofibrils, a two-dimensional liquid crystalline state and a three-dimensional hydrogel could be widely appreciated in various areas of nanobiotechnology including drug delivery and tissue engineering

In Situ Fibril Formation of κ‑Casein by External Stimuli within Multilayer Thin Films

We have developed the in situ fibrillation of κ-casein, employed as amyloid precursor, within multilayer films consisting of κ-casein and poly­(acrylic acid) (PAA) prepared by the layer-by-layer (LbL) deposition. The fibrillation of κ-casein within the multilayered films is strongly dependent on the extent of intermolecular interactions between κ-casein and PAA. When films constructed initially at pH 3 were heat treated at the same pH, κ-casein did not transform into fibrils. However, when the films were subjected to heat treatment at pH 5, κ-casein was transformed into fibrils within multilayer films due to weakened intermolecular interactions between κ-casein and PAA. We also noted that the multilayer film was swollen at pH 5 by the charge imbalance within the film, which we believe gives enough mobility for κ-caseins to form fibrils with adjacent κ-caseins within the multilayer. The fibrils were found to be uniformly distributed across the entire film thickness, and the aspect ratio as well as the number density of fibrils increased as a function of incubation time. The present study reveals a strategy to realize in situ nanocomposites within LbL multilayer films simply by triggering the formation of protein fibrils by controlling the intermolecular interactions between amyloid precursors and polyelectrolytes (PEs)

Additional file 2: Figure S2. of KBH-1, an herbal composition, improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats

Effect of KBH-1 on hepatic steatosis of HFD-induced obesity model. Animals were subdivided into 5 groups: ND, HFD, PC (treated with 200mg/kg of green tea extract), KBH-1 150mg/kg, and KBH-1 300 mg/kg. The body weight change of each group on HFD-induced obesity model. Data are expressed as the mean ± SEM. Significant differences from HFD group are indicated by *p < 0.05, **p < 0.01, or ***p < 0.005. (DOCX 55 kb

Additional file 1: Table S1. of KBH-1, an herbal composition, improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats

Blood chemical analysis on high-fat diet (HFD)-induced obesity rat model . HFDinduced rats show significant increase the level of GPT and LDL-C, and KBH-1 suppressed the level of GPT, LDL-C, and TG. (DOCX 16 kb

The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway

<div><p>The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments <i>in vitro</i> and <i>in vivo</i>. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells <i>in vitro</i>, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor (PPAR) γ, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal extract, as a potential anti-obesity therapeutic agent.</p></div

Effects of siRNA for AMPK on KBH-1-induced inhibition of adipocyte differentiation.

<p>3T3-L1 preadipocytes were induced to differentiate into mature adipocytes in the presence of KBH-1. Final concentration of 50 nM si RNA for AMPK (si-AMPK) was incubated with 3T3-L1 preadipocyte for 72 h, and then transfection medium was removed and cells were differentiated in the same condition as normal differentiation. <b>(A)</b> Lipid accumulation was measured using Oil Red O staining at a concentration of 10 μg/ml KBH-1 on day 7. (B) AMPK, ACC and PPARγ phosphorylation were measured using SDS-PAGE and immunoblotting. Bar graph (right panel) is the relative density after normalization to β-actin. Data are expressed as the mean ± SEM. Significant differences from the band of no si-AMPK treatment in the presence of KBH-1 are indicated by ** <i>p</i> < 0.01.</p

Effects of KBH-1 on AKT, ERK1/2, AMPK and ACC phosphorylation.

<p>3T3-L1 preadipocytes differentiated for 15, 30, 60 and 120 min in the absence or presence of 10 μg/ml KBH-1. AKT, ERK1/2, AMPK and ACC phosphorylation were measured using SDS-PAGE and immunoblotting. Bar graph (right panel) is the relative density after normalization to total form of each protein. Data are expressed as the mean ± SEM. Significant differences from each time-point control (no KBH-1 treatment) are indicated by *** <i>p</i> < 0.001. ■; 0 μg/ml KBH-1, □; 10 μg/ml KBH-1.</p

Receiver-operating characteristic (ROC) analyses for predicting renal pathology.

<p>ROC curves are shown according to the predictors and outcomes: (A) ROC curve for predicting interstitial fibrosis based on serum klotho, (B) ROC curve for predicting segmental sclerosis based on serum klotho, and (C) ROC curve for predicting foot process effacement based on the urinary klotho-to-creatinine ratio. Best cut-off values were presented as black circles and certain values (with specificity and sensitivity). AUC, area under the ROC curve; UKCR, urinary klotho-to-creatinine ratio.</p

Comparison of serum klotho levels in different groups for each pathology.

<p>Boxplots for serum klotho levels are presented according to the severity of each pathologic finding: (A) Interstitial fibrosis, (B) tubular atrophy, (C) segmental sclerosis of glomeruli, and (D) intimal thickening of arterial wall. ^*<i>P</i> < 0.01; ^**<i>P</i> < 0.001.</p

Association of serum klotho level with interstitial fibrosis and segmental sclerosis.

<p>Association of serum klotho level with interstitial fibrosis and segmental sclerosis.</p