Eggshell Membrane-Based Biotemplating of Mixed Hemimicelle/Admicelle as a Solid-Phase Extraction Adsorbent for Carcinogenic Polycyclic Aromatic Hydrocarbons

A new solid-phase extraction (SPE) format was demonstrated, based on eggshell membrane (ESM) templating of the mixed hemimicelle/admicelle of linear alkylbenzenesulfonates (LAS) as an adsorbent for the enrichment of carcinogenic polycyclic aromatic hydrocarbons (PAHs) in environmental aqueous samples. The LAS mixed hemimicelle/admicelle formation and SPE of the target PAHs were conducted simultaneously by adding the organic target and LAS through a column filled with 500 mg of ESM. The effect of various factors, including LAS concentration, solution pH, ionic strength, and humic acid concentration on the recoveries of PAHs were investigated and optimized. The results showed that LAS concentration and solution pH had obvious effect on extraction of PAHs, and the recoveries of PAHs compounds decreased in the presence of salt and humic acid. Under the optimized analytical conditions, the present method could respond down to 0.1–8.6 ng/L PAHs with a linear calibration ranging from 0.02 to 10 μg/L, showing a good PAHs enrichment ability with high sensitivity. The developed method was used satisfactorily for the detection of PAHs in environmental water samples. The mixed hemimicelle/admicelle adsorbent exhibited high extraction efficiency to PAHs and good selectivity with respect to natural organic matter and was advantageous over commercial C₁₈ adsorbent, for example, high extraction yield, high breakthrough volume, and easy regeneration

Inositol polyphosphate-4-phosphatase type II plays critical roles in the modulation of cadherin-mediated adhesion dynamics of pancreatic ductal adenocarcinomas

<p>The inositol polyphosphate-4-phosphatase type II (INPP4B) has been mostly proposed to act as a tumor suppressor whose expression is frequently dysregulated in numerous human cancers. To date, little is unveiled about whether and how INPP4B will exert its tumor suppressive function on the turnover of cadherin-based cell-cell adhesion system in pancreatic ductal adenocarcinomas (PDACs) <i>in vitro</i>. Here we provide the evidence that INPP4B manipulates cadherin switch in certain PDAC cell lines through a phosphorylated AKT-inactivation manner. The knockdown of INPP4B in AsPC-1 results in a more invasive phenotype, and overexpression of it in PANC-1 leads to partial reversion of mesenchymal status and impediment of <i>in vitro</i> invasion but not migration. More importantly, E-cadherin (Ecad) is enriched in the early and sorting endosomes containing INPP4B by which its recycling rather than degradation is enabled. Immunohistochemical analysis of 39 operatively resected PDAC specimens reveals it is poorly differentiated, non-cohesive ones in which the INPP4B and Ecad are partially or completely compromised in expression. We therefore identify INPP4B as an tumor suppressor in PDAC which attenuates AKT activation and participates in preservation of Ecad in endocytic pool and cellular membrane.</p

Isolation, purification, and biological activities of polysaccharides from <i>Amorpha fruticosa</i> flowers

The extraction, isolation, structural characterisation and biological activities of polysaccharides from Amorpha fruticosa flowers were investigated. First, the crude polysaccharide AFP was extracted, and two major purified polysaccharide fractions AFP-2 and AFP-3 were isolated. The molecular weight and monosaccharide compositions of AFP-2 and AFP-3 were determined. Then the antioxidant activities of AFP, AFP-2 and AFP-3 were assessed by DPPH radical, β-Carotene bleaching and hydroxyl radical assays. All three tested polysaccharides showed good antioxidant activity while AFP was the strongest one. The study also showed that AFP, AFP-2 and AFP-3 have good tyrosinase inhibition, moisture absorption and retention activities. The results will provide a helpful reference for the application of polysaccharide from Amorpha fruticosa flowers as a natural cosmetic ingredient.</p

Fat body and cardiomyocyte Mtp regulate systemic lipid metabolism differently on normal food diet and high fat diet.

<p>(A–C) Whole-body TG levels of newly-eclosed flies with <i>Gal4</i> drivers only (controls) or with fat body-specific knockdown of <i>mtp</i> using <i>R4-Gal4</i> (A), <i>ppl-Gal4</i> (B), or <i>Lsp2-Gal4</i> (C) on NFD (blue) or HFD (red). (D-E) Whole-body TG levels of newly-eclosed flies with <i>Gal4</i> drivers only (controls) or with cardiomyocyte-specific knockdown of <i>mtp</i> using <i>Hand-Gal4</i> (D), or <i>TinC-Gal4</i> (E) on NFD (blue) or HFD (red). (F) Whole-body TG levels of newly-eclosed flies with <i>Gal4</i> drivers only (controls) or with muscle-specific knockdown of <i>mtp</i> using <i>Mhc-Gal4</i> on NFD (blue) or HFD (red). In all cases, TG levels (μg/μl) were normalized to total protein (μg/μl). Results are expressed as the fold change in whole fly normalized TG compared with that of the <i>Gal4</i> control flies on NFD (set to 1.0). Results are the mean ± SEM of the indicated number of flies (N) analyzed over at least 5 independent experiments. <i>P</i>-values are from Student’s <i>t</i>-test and are between <i>Gal4</i> control and <i>Gal4</i>-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype.</p

Inhibition of Mtp or apoLpp in fat body or cardiomyocytes promotes intestinal lipid accumulation on normal food diet and high fat diet.

<p>(A-C’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on NFD. (A, A’), control larvae (<i>ppl-Gal4</i>); (B, B’), larvae with fat body-specific KD of <i>mtp</i> (<i>ppl-Gal4>mtp</i>^<i>RNAi</i>); and (C, C’), larvae with fat body-specific KD of <i>apoLpp</i> (<i>ppl-Gal4>apoLpp</i>^<i>RNAi</i>). (A, B, C) Lower magnification (10X) images with scale bars representing 100 μm. (A’, B’, C’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes. (D-F’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on HFD. (D, D’), control larvae (<i>ppl-Gal4</i>); (E, E’), larvae with fat body-specific KD of <i>mtp</i> (<i>ppl-Gal4>mtp</i>^<i>RNAi</i>); and (F, F’), larvae with fat body-specific KD of <i>apoLpp</i> (<i>ppl-Gal4>apoLpp</i>^<i>RNAi</i>). (D, E, F) Lower magnification (10X) images with scale bars representing 100 μm. (D’, E’, F’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes. (G-I’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on NFD. (G, G’), control larvae (<i>Hand-Gal4</i>); (H, H’), larvae with cardiomyocyte-specific KD of <i>mtp</i> (<i>Hand-Gal4>mtp</i>^<i>RNAi</i>); and (I, I’), larvae with cardiomyocyte-specific KD of <i>apoLpp</i> (<i>Hand-Gal4>apoLpp</i>^<i>RNAi</i>). (G, H, I) Lower magnification (10X) images with scale bars representing 100 μm. (G’, H’, I’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes. (J-L’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on HFD. (J, J’), control larvae (<i>Hand-Gal4</i>); (K, K’), larvae with cardiomyocyte-specific KD of <i>mtp</i> (<i>Hand-Gal4>mtp</i>^<i>RNAi</i>); and (L, L’), larvae with cardiomyocyte-specific KD of <i>apoLpp</i> (<i>Hand-Gal4>apoLpp</i>^<i>RNAi</i>). (J, K, L) Lower magnification (10X) images with scale bars representing 100 μm. (J’, K’, L’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes.</p

High fat diet alters relative apoLpp expression levels in the cardiomyocytes and fat body.

<p>(A, B) Relative mRNA levels of apoLpp in larval fat body (A) and larval hearts (B) on NFD and HFD. Results are expressed as the fold difference compared with the NFD condition. Values were normalized with <i>gapdh</i>. ***<i>p</i> < 0.001, by two-tailed paired <i>t</i>-test analyzed over 3 independent experiments. (C) Relative mRNA levels of apoLpp in in larval heart relative to larval fat body on HFD. Results are expressed as the fold difference. Values were normalized with gapdh. *<i>p</i> < 0.05, ***<i>p</i> < 0.001, by two-tailed paired <i>t</i>-test analyzed over 3 independent experiments. (D) Relative mRNA levels of apoLpp in larval heart relative to larval fat body on NFD. Results are expressed as the fold difference. Values were normalized with gapdh. *<i>p</i> < 0.05, ***<i>p</i> < 0.001, by two-tailed paired <i>t</i>-test analyzed over 3 independent experiments. (E-E”) Representative confocal images of apoLpp (E), Boca (E’), and apoLpp, Boca and DAPI (E”) in cardiomyocytes of the <i>Hand-Gal4</i> control third instar larvae on NFD. Scale bars represent 40 μm. Boca marks the endoplasmic reticulum and DAPI marks the nucleus in each cardiomyocyte. (F-F”) Representative confocal images of apoLpp (F), Boca (F’), and apoLpp, Boca and DAPI (F”) in cardiomyocytes of the <i>Hand-Gal4</i> control third instar larvae on HFD. White arrows in F and F” indicate the apoLpp puncta which reflect the presence of Lpp. Yellow arrows in F’ and F” indicate the endoplasmic reticulum marker Boca, and DAPI marks the nuclei in the cardiomyocytes. The magenta arrow in F” indicates the co-localization of apoLpp and Boca. Scale bars represent 40 μm. (G-G”) Representative confocal images of apoLpp (G), Boca (G’), and apoLpp, Boca and DAPI (G”) in cardiomyocytes of the <i>Hand-Gal4</i>-mediated <i>mtp</i> KD third instar larvae on HFD. White arrows in G and G” indicate the strong punctate staining of apoLpp which reflects the accumulation of Lpp. Yellow arrows in G’ and G” indicate the endoplasmic reticulum marker Boca, and DAPI marks the nuclei in the cardiomyocytes. Scale bars represent 40 μm.</p

Genetic screening identifies Mtp as a gene for determining systemic lipid metabolism.

<p>(A) Schematic of the results of the genetic screen showing the location of the <i>mtp</i> gene (blue symbol) on chromosome 2L. Red bars indicate deletions in the three deficiency lines investigated here: <i>Df(2L)ED1378</i>, <i>Df(2L)BSC333</i>, and <i>Df(2L)Exel7080</i>. (B) Whole-body TG levels of newly eclosed control (<i>w</i>^<i>1118</i>) flies and deficiency lines on NFD or HFD. For each genotype, a 1:1 ratio of males:females was analyzed. <i>P</i>-values are from Student’s <i>t</i>-test and are between control <i>w</i>^<i>1118</i> and deficiency lines within NFD or HFD, or between NFD and HFD for the same genotype. (C) Whole-body TG levels of newly eclosed control flies (<i>Arm-Gal4</i>), and flies with whole-body KD of <i>mtp</i> (<i>Arm-Gal4>mtp</i>^<i>RNAi</i>) on NFD and HFD. For each genotype, a 1:1 ratio of males:females was analyzed. <i>P</i>-values are from Student’s <i>t</i>-test and are between <i>Gal4</i> control and <i>Gal4</i>-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype. (D) Whole-body TG levels of newly eclosed control flies (<i>Da-Gal4</i>), and flies with whole-body KD of <i>mtp</i> (<i>Da-Gal4>mtp</i>^<i>RNAi</i>) on NFD and HFD. For each genotype, a 1:1 ratio of males:females was analyzed. <i>P</i>-values are from Student’s <i>t</i>-test and are between <i>Gal4</i> control and <i>Gal4</i>-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype. In (B), (C) and (D), TG levels (μg/μl) were normalized to total protein (μg/μl). Results are expressed as the fold change in whole fly normalized TG compared with that of the wild-type <i>w</i>^<i>1118</i> or <i>Gal4</i> control flies on NFD (set to 1.0). Results are the mean ± SEM of the indicated number of flies (N) analyzed over at least 5 independent experiments. (E) Colorimetric assay of food intake in control <i>w</i>^<i>1118</i> third instar larvae and third instar larvae with <i>Gal4</i> driver only (<i>Arm-Gal4</i>) or with whole body knockdown of <i>mtp</i> using <i>Arm-Gal4</i> (<i>Arm-Gal4>mtp</i>^<i>RNAi</i>). Results are the mean ± SEM of the indicated number of larvae (N) analyzed over 3 independent experiments. <i>P</i>-values are from Student’s <i>t</i>-test.</p

Fat body and cardiomyocyte apoLpp regulate systemic lipid metabolism differently on normal food diet and high fat diet.

<p>(A) Whole-body TG levels of newly-eclosed flies with <i>Gal4</i> driver only (control) or with fat body-specific knockdown of <i>apoLpp</i> using <i>ppl-Gal4</i> (A) on NFD (blue) or HFD (red). (B, C) Whole-body TG levels of newly-eclosed flies with <i>Gal4</i> drivers only (controls) or with cardiomyocyte-specific knockdown of <i>apoLpp</i> using <i>Hand-Gal4</i> (B), or <i>TinC-Gal4</i> (C) on NFD (blue) or HFD (red). (D) RT-PCR analysis of <i>mtp</i> mRNA levels in whole flies with <i>Arm-Gal4</i> driver only (control) or with <i>Arm-Gal4</i>-induced overexpression of full-length <i>mtp</i> (<i>mtp</i>^<i>+</i>). Actin serves as an internal control. (E) Whole-body TG levels of newly-eclosed flies with <i>Gal4</i> driver only (control), with cardiomyocyte-specific overexpression of <i>mtp</i>^<i>+</i> using <i>TinC-Gal4</i>, or with cardiomyocyte-specific overexpression of <i>mtp</i>^<i>+</i> and <i>apoLppRNAi</i> using <i>TinC-Gal4</i> on NFD (blue) or HFD (red). In A-C and E, TG levels (μg/μl) were normalized to total protein (μg/μl). Results are expressed as the fold change in whole fly normalized TG compared with that of the <i>Gal4</i> control flies on NFD (set to 1.0). Results are the mean ± SEM of the indicated number of flies (N) analyzed over at least 5 independent experiments. <i>P</i>-values are from Student’s <i>t</i>-test and are between <i>Gal4</i> control and <i>Gal4</i>-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype.</p

A model depicting the relative contributions of Lpp derived from the fat body and cardiomyocytes in controlling systemic lipid metabolism on NFD and HFD.

<p>(A) On NFD, Lpp derived from the cardiomyocytes play an equally important role as Lpp derived from the fat body in systemic lipid homeostasis maintenance. Higher levels of Mtp and apoLpp (blue threads) are present in the fat body than in cardiomyocytes on NFD. Both the cardiomyocyte- and fat body-derived Lpp are recruited to the intestine where they promote the uptake of dietary lipids from the enterocytes and transport the dietary lipids to peripheral tissues for energy production or for storage in the fat body. (B) HFD induces an upregulation of the relative expression of Mtp and apoLpp in the cardiomyocytes (red arrows) and downregulation of their relative expression in the fat body (green arrows), culminating in higher levels of Mtp (bold) and apoLpp (blue threads) in the cardiomyocytes than in the fat body. This could underlie the predominant role of the cardiomyocyte-derived Lpp in the determining of lipid metabolic responses to HFD (thick versus broken arrows, B).</p