A genome-wide survey of copy number variation regions in various chicken breeds by array comparative genomic hybridization method

The discovery of copy number variation (CNV) in the genome has provided new insight into genomic polymorphism. Studies with chickens have identified a number of large CNV segments using a 385k comparative genomic hybridization (CGH) chip (mean length >140 kb). We present a detailed CNV map for local Chinese chicken breeds and commercial chicken lines using an Agilent 400k array CGH platform with custom-designed probes. We identified a total of 130 copy number variation regions (CNVRs; mean length = 25.70 kb). Of these, 104 (80.0%) were novel segments reported for the first time in chickens. Among the 104 novel CNVRs, 56 (53.8%) of the segments were non-coding sequences, 65 (62.5%) showed the gain of DNA and 40 (38.5%) showed the loss of DNA (one locus showed both loss and gain). Overlapping with the formal selective sweep data and the quantitative trait loci data, we identified four loci that might be considered to be high-confidence selective segments that arose during the domestication of chickens. Compared with the CNVRs reported previously, genes for the positive regulation of phospholipase A2 activity were discovered to be significantly over-represented in the novel CNVRs reported here by gene ontology analysis. Availability of our results should facilitate further research in the study of the genetic variability in chicken breeds.     

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.     

Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing

Despite the large number of receptor-like kinases (RLKs) in plants, few of their specific ligands are known. Ligand fishing is one of the most challenging post-genomic technologies. Here, we report a strategy for functional immobilization of plant RLKs on microbeads via covalent linkage. Because of the high density of immobilized RLKs, ligand-receptor interaction can be visualized at high sensitivity using fluorescent-labeled ligands under the confocal laser scanning microscope. Moreover, using a receptor-based affinity chromatography system with RLK microbeads, the ligand of the receptor was directly retrieved at high purity from complex natural samples. Our RLK microbeads and receptor-based ligand fishing approach is a feasible alternative to conventional forward genetics and bioassay-based biochemical purification for identification of novel ligand-receptor pairs in plants.     

Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer

Butyrate producers constitute an important bacterial group in the human large intestine. Butyryl-CoA is formed from two molecules of acetyl-CoA in a process resembling beta-oxidation in reverse. Three different arrangements of the six genes coding for this pathway have been found in low mol% G+C-content gram-positive human colonic bacteria using DNA sequencing and degenerate PCR. Gene arrangements were strongly conserved within phylogenetic groups defined by 16S rRNA gene sequence relationships. In the case of one of the genes, encoding beta-hydroxybutyryl-CoA dehydrogenase, however, sequence relationships were strongly suggestive of horizontal gene transfer between lineages. The newly identified gene for butyryl-CoA CoA-transferase, which performs the final step in butyrate formation in most known human colonic bacteria, was not closely linked to these central pathway genes.     

Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1)

Heat shock protein 20 (HSP20) has cardioprotective qualities, which are triggered by PKA phosphorylation. PKD1 is also a binding partner for HSP20, and this prompted us to investigate whether the chaperone was a substrate for PKD1. We delineate the PKD1 binding sites on HSP20 and show for the first time HSP20 is a substrate for PKD1. Phosphorylation of HSP20 by PKD1 is diminished by pharmacological or siRNA reduction of PKD1 activity and is enhanced following PKD1 activation. Our results suggest that both PKA and PKD1 can both phosphorylate HSP20 on serine 16 but that PKA is the most dominant. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Proteome array identification of bioactive soluble proteins/peptides in Matrigel: relevance to stem cell responses

Matrigel and similar commercial products are extracts of the Engelbreth-Holm–Swarm sarcoma that provide a basement-membrane-like attachment substrate or gel that is used to grow cells on or in, respectively. To ascertain further what proteins may be present in Matrigel, besides its major basement-membrane constituents, an analysis of the expressed liquid of gelled Matrigel was performed using proteome array technology. Among the growth factors/cytokines assayed, high positive detection was found for IGFBP1, IGFBP3, LIF, platelet factor 4, PlGF-2, and VEGF; moderate reactivity was found for cyr61, IGFBP2, IGFBP6, IL-1ra, and NOV; and low, but detectable, responses occurred for aFGF, IL-13, IL-23, M-CSF, and VEGF-B. Among the chemokines assayed, high positive detection was found for MIG and serpin E1; moderate reactivity was found for IP-10, MCP-1, and MCP-5, and low, but detectable, responses occurred for CXCL16, I-TAC, and MIP-1α. Among the other biologically active proteins assayed, high positive detection was found for adiponectin, C5a, endocan, lipocalin-2, sICAM-1, MMP-3, and TIMP-1; moderate reactivity was found for C-reactive protein, coagulation factor III, endoglin, endostatin/collagen XVIII, endothelin-1, ICAM-1, MMP-9, osteopontin, pentraxin-3, and RANTES; and low, but detectable, responses occurred for fetuin A, MMP-8, pentraxin-2, RBP4, resistin, and TIMP-4. The study found several growth factors, chemokines, and biologically active proteins not previously identified in Matrigel, and this may have significance to the interpretations of observed cellular responses when cells are grown on or in Matrigel.     

Parallel detection of ancient pathogens via array-based DNA capture

DNA capture coupled with next generation sequencing is highly suitable for the study of ancient pathogens. Screening for pathogens can, however, be meticulous when assays are restricted to the enrichment of single organisms, which is common practice. Here, we report on an array-based DNA capture screening technique for the parallel detection of nearly 100 pathogens that could have potentially left behind molecular signatures in preserved ancient tissues. We demonstrate the sensitivity of our method through evaluation of its performance with a library known to harbour ancient Mycobacterium leprae DNA. This rapid and economical technique will be highly useful for the identification of historical diseases that are difficult to characterize based on archaeological information alone.     

Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase

Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.