Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry

The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH‐MS to perform reproducible proteomic quantification of biopsy‐level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT‐SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3–0.2 mg). The data show that as few as 50 000 human cells and 0.2–0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH‐MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT‐SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.     

Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re‐establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full‐length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9‐week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell‐based gene therapy provides a new approach to cardiac regeneration.     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

A catalogue of Burmite inclusions

Burmite (Burmese amber) from the Hukawng Valley in northern Myanmar is a remarkable valuable and obviously the most important amber for studying terrestrial diversity in the mid-Cretaceous.The diversity of Burmite inclusions is very high and many new taxa were found,including new order,new family/subfamily,and new genus.Till the end of 2016,14 phyla,21 classes,65 orders,279 families,515 genera and 643 species of organisms are recorded,which are summized and complied in this catalogue.Among them,587 species are arthropods.In addtion,the specimens which can not be identified into species are also listed in the paper.The information on type specimens,other materials,host and deposition of types are provided.