Transcriptome Profiling of Whole Blood Cells Identifies PLEK2 and C1QB in Human Melanoma

Developing analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a "nucleic acid-rich" and "inflammatory cell-rich" information reservoir and represents systemic processes altered by the presence of cancer cells.We conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV) and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45(-) and CD45(+) populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively.The current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45(-) subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients for residual disease

A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation

© 2019 The Authors. Published by Springer. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1038/s41467-019-13149-7The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.This work was funded by the Novo Nordisk Foundation (NNF17CC0027852) and Danish National Research Foundation (DNRF116). J.C. and D.G.W. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001217), the UK Medical Research Council (FC001217), and the Wellcome Trust (FC001217). S.C. was supported by an SNSF Early Postdoc Mobility fellowship (P2ZHP3_164840) and a Long Term EMBO Postdoc fellowship (ALTF 511-2016), and L.S. and J.B.A. by the Independent Research Fund Denmark (DFF; Sapere Aude2 4183-00118B).Published versio

Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains.

We have isolated cya-lac operon and protein fusions in Escherichia coli K-12, and we used these to study the regulation of cya, the structural gene for adenylate cyclase. Data obtained from these fusion strains suggest that neither cyclic AMP (cAMP) nor the cAMP receptor protein plays a major role in transcriptional or translational regulation of cya expression. Modulation of intracellular cAMP concentrations elicited only weak repression of cya-lac fusion activity under conditions of high intracellular cAMP, relative to fusion activity under conditions of low intracellular cAMP. The functional cAMP receptor protein was required for this effect. Incorporation of delta crp into cya-lac fusion strains did not affect fusion expression in glucose-grown cells as compared with similarly cultured isogenic crp+ strains. Furthermore, 20 independently obtained mutants derived from a cya-lacZ protein fusion strain exhibiting a weak Lac+ phenotype were isolated, and it was determined that the mutants had beta-galactosidase activities ranging from 2- to 77-fold greater than those of the parental strain. None of the mutations responsible for this increase in fusion activity map in the crp locus. We used these mutants to aid in the identification of a 160,000-dalton cya-lacZ hybrid protein. Finally, chromosome mobilization experiments, using cya-lac fusion strains, allowed us to infer a clockwise direction of transcription for the cya gene relative to the standard E. coli genetic map

Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope.

An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently. Consequently, such a strain is defective for the utilization of maltose as a sole carbon source. We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein. A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E. coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map. Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope. Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur. Our data suggest that proper assembly of a complex is required for efficient export of E. coli envelope proteins to their various extracytoplasmic compartments

Export and processing of MalE-LacZ hybrid proteins in Escherichia coli.

Five classes of MalE-LacZ hybrid proteins have previously been characterized. These proteins differ in the amount of the maltose-binding protein (MBP) that is attached to beta-galactosidase. Although none of these proteins is secreted into the periplasm, the four larger classes of hybrid proteins, those that include an intact MBP signal peptide, are inserted into the cytoplasmic membrane, suggesting that the secretion process has at least been initiated. In this study, we demonstrated that some portion of the four larger hybrid proteins can be translocated across the cytoplasmic membrane, thus permitting processing of the signal peptide. We have found that hybrid proteins that include only a small portion of the mature MBP are inefficiently recognized as exported proteins, and translocation and processing of these appear to be relatively slow, posttranslational events. In marked contrast, hybrid proteins that include a substantial portion of the mature MBP are efficiently recognized, and translocation and processing of these occur very rapidly, possibly cotranslationally. Our results complement other studies and very strongly suggest a role for the mature MBP in the export process

Isolation of yeast mutants defective in protein targeting to the vacuole.

We have constructed a PRC1-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that "mislocalize" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent defects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic

Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli.

We have previously described the construction in Escherichia coli K-12 of a hybrid plasmid colony bank of Treponema pallidum (Nichols strain) genomic DNA. By screening a portion of this bank with an in situ immunoassay, we identified six E. coli clones that express T. pallidum antigens. In this study, the recombinant plasmids from each of these clones have been analyzed in E. coli maxicells and have been found to encode a number of proteins that are not of vector pBR322 origin and are, therefore, of treponemal origin. In each case, several of these proteins can be specifically precipitated from solubilized maxicell extracts by high-titer experimental rabbit syphilitic serum. Certain of these proteins are also precipitated by high-titer latent human syphilitic sera (HSS). The T. pallidum DNA inserts in these plasmids range in size from 6.2 to 14 kilobase pairs, and from the restriction patterns of the inserts and the protein profiles generated by each plasmid in maxicells, it is apparent that we have recovered a total of four unique clones from our colony bank. Recombinant plasmids pLVS3 and pLVS5 were of particular interest. Plasmid pLVS3 encodes three major protein antigens with molecular weights of 39,000, 35,000, and 25,000. These three proteins, which were not recognized by pooled normal human sera, were efficiently precipitated by most secondary HSS, latent HSS, and late HSS tested. These proteins were also precipitated, although somewhat inefficiently, by most primary HSS tested. Plasmid pLVS5 encodes a major protein antigen with a molecular weight of 32,000 and several minor protein antigens that, although efficiently precipitated by experimental rabbit syphilitic serum, were generally not recognized by the various HSS tested. Evidence is presented indicating that the protein antigens encoded by plasmids pLVS3 and pLVS5 are specific for pathogenic treponemal species. We have also demonstrated that immunoglobulin G antibodies directed against these protein antigens can be detected in rabbits experimentally infected with T. pallidum Nichols as early as 11 days postinfection