A miniaturized sandwich immunoassay platform for the detection of protein-protein interactions

<p>Abstract</p> <p>Background</p> <p>Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.</p> <p>Results</p> <p>Based on anti-FLAG antibody spotted aldehyde slides, MSIP enables simple, rapid and large scale detection of PPIs by fluorescent labeling anti-myc antibody. By analyzing well-known interacting and non-interacting protein pairs, MSIP was demonstrated to be highly accurate and reproducible. Compared to traditional resin-based coIP, MSIP results in higher sensitivity and enhanced throughput, with the additional benefit of digital read-outs. In addition, MSIP was shown to be a highly useful validation platform to confirm PPI candidates that have been identified from yeast two hybrid systems.</p> <p>Conclusions</p> <p>In conclusion, MSIP is proved to be a simple, cost-saving and highly efficient technique for the comprehensive study of PPIs.</p

Novel compounds in fruits of coriander (Coşkuner & Karababa) with anti-inflammatory activity

© 2020 Coriander, Coriandrum Sativum L., is one of the commonest food and medicinal plants in many countries, but its chemical ingredients and pivotal role in anti-inflammatory activity have not been fully explored. The present study aimed to identify new compounds in the fruits of coriander and explore their anti-inflammatory activity. The compounds were isolated by chromatographic seperations and identified using spectroscopic and spectrometric methods. RAW264.7 macrophage cells were used to detect the anti-inflammatory activity of the compounds via Griess assay, western blotting, ELISA, and flow cytometry methods. The study resulted in the discovery of four new compounds, which were identified as: 4α-(furo[2,3-d]pyrimidin-6′-ylmethyl)-9α-propylnonolactone (1), 4-(formyloxy)-4-(6′-methylcyclohex-1-en-1-yl)butanoate(2), (7α,8α)-3α-hydroxyl-12,13α-dimethyl-5(6)-en-bicyclo[5,3,0]caprolactone (3), 7-methoxy-4-methyl-5,6-dihydro-7H-(2-hydroxypropan-2-yl)furo[2,3-f] coumarin (4). Compound 3 showed the highest anti-inflammatory activity with IC50 of 6.25 μM for an inhibitory effect on nitrite oxide (NO) level. In addition, compound 3 decreased the lipopolysaccharides-stimulated generations of ROS and the inflammatory cytokines (IL-6 and TNF-α). Mechanism exploration indicated that compound 3 suppressed inflammatory mediators’ expression, like iNOS and COX-2. Furthermore, the NF-κB and MAPK pathways were involved in the anti-inflammatory process of compound 3

Genetic backgrounds determine brown remodeling of white fat in rodents

Objective: Genetic background largely contributes to the complexity of metabolic responses and dysfunctions. Induction of brown adipose features in white fat, known as brown remodeling, has been appreciated as a promising strategy to offset the positive energy balance in obesity and further to improve metabolism. Here we address the effects of genetic background on this process. Methods: We investigated browning remodeling in a depot-specific manner by comparing the response of C57BL/6J, 129/Sv and FVB/NJ mouse strains to cold. Results: Surprisingly, 129/Sv and FVB/NJ mice showed distinct brown remodeling features despite their similar resistance to metabolic disorders in comparison to the obesity-prone C57BL/6J mice. FVB/NJ mice demonstrated a preference of brown remodeling in inguinal subcutaneous white adipose tissue (iWAT), whereas 129/Sv mice displayed robust brown remodeling in visceral epididymal fat (eWAT). We further compared gene expression in different depots by RNA-sequencing and identified Hoxc10 as a novel “brake” of brown remodeling in iWAT. Conclusion: Rodent genetic background determines the brown remodeling of different white fat depots. This study provides new insights into the role of genetic variation in fat remodeling in susceptibility to metabolic diseases. Author Video: Author Video Watch what authors say about their articles Keywords: Genetic background, White adipose tissue, Brown remodeling, Hoxc10, Cold exposure, Browning brak

Antischistosomal Properties of Hederacolchiside A1 Isolated from Pulsatilla chinensis

Background: Schistosomiasis is a major neglected disease for which the current control strategy involves mass treatment with praziquantel, the only available drug. Hence, there is an urgent need to develop new antischistosomal compounds. Methods: The antischistosomal activity of hederacolchiside A1 (HSA) were determined by total or female worm burden reductions in mice harboring Schistosoma japonicum or S. mansoni. Pathology parameters were detected on HSA against 1-day-old S. japonicum-harboring mice. Moreover, we confirmed the antischistosomal effect of HSA on newly transformed schistosomula (NTS) of S. japonicum in vitro. Results: HSA, a natural product isolated from Pulsatilla chinensis (Bunge) Regel, was initially corroborated to possess promising antischistosomal properties. We demonstrated that HSA had high activity against S. japonicum and S. mansoni less in 11 days old parasites harbored in mice. The antischistosomal effect was even more than the currently used drugs, praziquantel, and artesunate. Furthermore, HSA could ameliorate the pathology parameters in mice harboring 1-day-old juvenile S. japonicum. We also confirmed that HSA-mediated antischistosomal activity is partly due to the morphological changes in the tegument system when NTS are exposed to HSA. Conclusions: HSA may have great potential to be an antischistosomal agent for further research

Anti-inflammatory and cytotoxic withanolides from Physalis minima

Six undescribed withanolides were isolated and characterized during the investigation of anti-inflammatory and cytotoxic constituents from the whole plants of Physalis minima L. Their structures were elucidated by extensive spectroscopic analyses (IR, UV, HR-ESI-MS, 1D-NMR, and 2D-NMR), and their anti-inflammatory and cytotoxic activities were evaluated in vitro. All the compounds exhibited anti-inflammatory ability via inhibiting the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. Moderate cytotoxic activities against A549 lung adenocarcinoma cells, SMMC-7721 hepatic carcinoma cells and MCF-7 breast cancer cells with IC50 values in the range of 40.01-82.17 mu M were observed for these withanolides

Locally Induced Adipose Tissue Browning by Microneedle Patch for Obesity Treatment

Obesity is one of the most serious public health problems in the 21st century that may lead to many comorbidities such as type-2 diabetes, cardiovascular diseases, and cancer. Current treatments toward obesity including diet, physical exercise, pharmacological therapy, as well as surgeries are always associated with low effectiveness or undesired systematical side effects. In order to enhance treatment efficiency with minimized side effects, we developed a transcutaneous browning agent patch to locally induce adipose tissue transformation. This microneedle-based patch can effectively deliver browning agents to the subcutaneous adipocytes in a sustained manner and switch on the “browning” at the targeted region. It is demonstrated that this patch reduces treated fat pad size, increases whole body energy expenditure, and improves type-2 diabetes <i>in vivo</i> in a diet-induced obesity mouse model

New Triterpenoid Saponins from Ilex cornuta and Their Protective Effects against H₂O₂‑Induced Myocardial Cell Injury

Five new triterpenoid saponins, <b>1</b>–<b>5</b>, together with 10 known ones, <b>6</b>–<b>15</b> were isolated from the aerial parts of Ilex cornuta. The structures of compounds <b>1</b>–<b>5</b> were determined as 3β-<i>O</i>-α-l-arabinopyranosyl-19α,23-dihydroxy-20α-urs-12-en-28-oic acid 28-<i>O</i>-β-d-glucopyranosyl ester, <b>1</b>; 3β-<i>O</i>-β-d-glucopyranosyl-(1→2)-α-l-arabinopyranosyl-19-hydroxy-20α-urs-12-en-28-oic acid 28-<i>O</i>-β-d-glucopyranosyl ester, <b>2</b>; 19α,23-dihydroxyurs-12-en-28-oic acid 3β-<i>O</i>-β-d-glucuronopyranoside-6-<i>O</i>-methyl ester, <b>3</b>; 19α,23-dihydroxyurs-12-en-28-oic acid 3β-<i>O</i>-[β-d-glucuronopyranoside-6-<i>O</i>-methyl ester]-28-<i>O</i>-β-d-glucopyranosyl ester, <b>4</b>; and 3β-<i>O</i>-[α-l-arabinopyranosyl-(1→2)-β-d-glucuronic acid]-oleanolic acid 28-<i>O</i>-β-d-glucopyranosyl ester, <b>5</b>, on the basis of spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR) and chemical reactions. Protective effects of compounds <b>1</b>–<b>15</b> were tested against H₂O₂-induced H9c2 cardiomyocyte injury, and the data showed that compounds <b>1</b>, <b>4</b>, <b>6</b>, and <b>13</b> had significant cell-protective effects. No significant DPPH radical scavenging activity was observed for compounds <b>1</b>–<b>15</b>

Simultaneous Purification of Pulchinenoside B₄ and B₅ from <i>Pulsatilla chinensis</i> Using Macroporous Resin and Preparative High Performance Liquid Chromatography

Pulchinenoside B₄ and B₅ (PB₄, PB₅) are two major triterpenoid saponins existing in the roots of <i>Pulsatilla chinensis</i> (Bunge) Regel. In this study, a systematic preparative process was developed for the simultaneous purification of PB₄ and PB₅ from the herb. The performance and separation characteristics of nine types of macroporous resins were critically evaluated. Static absorption/desorption experiments revealed that LX17 belonging to the polyacrylate class possessed superior separation properties. Further dynamic absorption/desorption experiments on LX17 column were conducted to obtain the optimal parameters. To obtain both compounds with high purity, a second stage procedure was developed using preparative reversed-phase high performance liquid chromatography with a dynamic axial compression column system. The separation process was high-efficiency and low-cost, which indicated potential for industrial applications

Adipsin promotes bone marrow adiposity by priming mesenchymal stem cells

Background: Marrow adipose tissue (MAT) has been shown to be vital for regulating metabolism and maintaining skeletal homeostasis in the bone marrow (BM) niche. As a reflection of BM remodeling, MAT is highly responsive to nutrient fluctuations, hormonal changes, and metabolic disturbances such as obesity and diabetes mellitus. Expansion of MAT has also been strongly associated with bone loss in mice and humans. However, the regulation of BM plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling. Methods: We studied deletion of Adipsin, and its downstream effector, C3, in C57BL/6 mice as well as the bone-protected PPARγ constitutive deacetylation 2KR mice to assess BM plasticity. The mice were challenged with thiazolidinedione treatment, calorie restriction, or aging to induce bone loss and MAT expansion. Analysis of bone mineral density and marrow adiposity was performed using a μCT scanner and by RNA analysis to assess adipocyte and osteoblast markers. For in vitro studies, primary bone marrow stromal cells were isolated and subjected to osteoblastogenic or adipogenic differentiation or chemical treatment followed by morphological and molecular analyses. Clinical data was obtained from samples of a previous clinical trial of fasting and high-calorie diet in healthy human volunteers. Results: We show that Adipsin is the most upregulated adipokine during MAT expansion in mice and humans in a PPARγ acetylation-dependent manner. Genetic ablation of Adipsin in mice specifically inhibited MAT expansion but not peripheral adipose depots, and improved bone mass during calorie restriction, thiazolidinedione treatment, and aging. These effects were mediated through its downstream effector, complement component C3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through inhibiting Wnt/β-catenin signaling. Conclusions: Adipsin promotes new adipocyte formation and affects skeletal remodeling in the BM niche. Our study reveals a novel mechanism whereby the BM sustains its own plasticity through paracrine and endocrine actions of a unique adipokine. Funding: This work was supported by the National Institutes of Health T32DK007328 (NA), F31DK124926 (NA), R01DK121140 (JCL), R01AR068970 (BZ), R01AR071463 (BZ), R01DK112943 (LQ), R24DK092759 (CJR), and P01HL087123 (LQ)