Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism

Microservice Transition and its Granularity Problem: A Systematic Mapping Study

Microservices have gained wide recognition and acceptance in software industries as an emerging architectural style for autonomic, scalable, and more reliable computing. The transition to microservices has been highly motivated by the need for better alignment of technical design decisions with improving value potentials of architectures. Despite microservices' popularity, research still lacks disciplined understanding of transition and consensus on the principles and activities underlying "micro-ing" architectures. In this paper, we report on a systematic mapping study that consolidates various views, approaches and activities that commonly assist in the transition to microservices. The study aims to provide a better understanding of the transition; it also contributes a working definition of the transition and technical activities underlying it. We term the transition and technical activities leading to microservice architectures as microservitization. We then shed light on a fundamental problem of microservitization: microservice granularity and reasoning about its adaptation as first-class entities. This study reviews state-of-the-art and -practice related to reasoning about microservice granularity; it reviews modelling approaches, aspects considered, guidelines and processes used to reason about microservice granularity. This study identifies opportunities for future research and development related to reasoning about microservice granularity.Comment: 36 pages including references, 6 figures, and 3 table

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Background Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Results Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring. Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: A multi-center evaluation

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.This work was supported by the National Collaborative Research Infrastructure (NCRIS) via the Australian Phenomics Network (APN) (to Gaetan Burgio and Paul Thomas), by an Institutional Development Award (PI: Shelley Smith) P20GM103471 (to CBG, RMQ, DWH, JDE, and RR), by NIGMS 1P30GM110768-01 and P30CA036727 (as part of support to University of Nebraska Mouse Genome Engineering and DNA Sequencing Cores), the British Heart Foundation FS12-57, FS12/57/29717, and CH/13/2/30154 and the program grant RG/15/12/31616 (to Kathryn Hentges and Bernard Keavney), the Wellcome Trust grants 107849/Z/ 15/Z, 097820/Z11/B, and 105610/Z/14/Z, the Medical Research Council MR/ N029992/1 (to DB and CBL), the National BioResource Project of Ministry of Education, Culture, Sports, Science and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan, the Canadian Institutes of Health Research MOP#142452 (MCB and LJM). LJM is a member of the Research Centre of the McGill University Health Centre which is supported in part by FQRS. Dr. William Thompson was supported by the Indiana Clinical and Translational Sciences Institute, funded in part by grant #UL1 TR001108 from the National Institute of Health (NIH), National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. KC Kent Lloyd is supported by the NIH (UM1OD023221), and work contributed by staff from the UC Davis Mouse Biology Program (MBP) is supported by a grant from the American College of Laboratory Animal Medicine. The work contributed from Xiande Liu, Chad Smith, Eric Jonasch, Xuesong Zhang, and Jan ParkerThornburg is supported by the NIH under the award number P30CA16672 (XL, CS, EJ, XZ, JPT) and R50CA211121 (JPT). Joseph Miano is supported by the NIH under the award number HL138987. R Sedlacek was supported by LM2015040 (Czech Centre for Phenogenomics), CZ.1.05/1.1.00/02.0109 (BIOCEV), and CZ.1.05/2.1.00/19.0395 by the Ministry of Education, Youth and Sports (MEYS) and by Academy of Sciences of the Czech Republic (RVO 68378050). David Ray was supported by a Wellcome Trust Investigator (107849/Z/15/Z) and the Medical Research Council (MR/P011853/1 and MR/P023576/) grants. Andrew Loudon was supported by a Wellcome Trust Investigator (107849/Z/15/Z), Biotechnology and Biological Sciences Research Council (BB/N015584/1), Medical Research Council (MR/P023576/1). The work contributed from Gloria Lopez-Castejon is supported by the Wellcome Trust (104192/Z/14/Z) and the Royal Society. Pilar Alcaide was supported by the NIH (HL 123658). The work contributed from Surinder K. Batra is supported by the NIH under the award number P01 CA217798

Renal Thrombotic Microangiopathy in Mice with Combined Deletion of Endocytic Recycling Regulators EHD3 and EHD4

Eps15 Homology Domain-containing 3 (EHD3), a member of the EHD protein family that regulates endocytic recycling, is the first protein reported to be specifically expressed in the glomerular endothelium in the kidney; therefore we generated Ehd3–/– mice and assessed renal development and pathology. Ehd3–/– animals showed no overt defects, and exhibited no proteinuria or glomerular pathology. However, as the expression of EHD4, a related family member, was elevated in the glomerular endothelium of Ehd3–/– mice and suggested functional compensation, we generated and analyzed Ehd3–/–; Ehd4–/– mice. These mice were smaller, possessed smaller and paler kidneys, were proteinuric and died between 3–24 weeks of age. Detailed analyses of Ehd3–/–; Ehd4–/– kidneys demonstrated thrombotic microangiopathy (TMA)-like glomerular lesions including thickening and duplication of glomerular basement membrane, endothelial swelling and loss of fenestrations. Other changes included segmental podocyte foot process effacement, mesangial interposition, and abnormal podocytic and mesangial marker expression. The glomerular lesions observed were strikingly similar to those seen in human pre-eclampsia and mouse models of reduced VEGF expression. As altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis was observed in the absence of EHD3 and EHD4, we propose that EHD-mediated endocytic traffic of key surface receptors such as VEGFR2 is essential for physiological control of glomerular function. Furthermore, Ehd3–/–; Ehd4–/– mice provide a unique model to elucidate mechanisms of glomerular endothelial injury which is observed in a wide variety of human renal and extra-renal diseases

Solid-​phase synthesis of the hexapeptide sequence of the active site of triosephosphate isomerase

Solid phase synthesis of the protected hexampeptide, Z-​Ala-​Tyr-​Glu(OCH2Ph)​-​Pro-​Val-​Trp-​OCH2Ph (II, Z = PhCH2O2C)​, was reported. This peptide was released from the peptide polymer by transesterification with PhCH2OH in Et3N at 60°. Hydrogenation of II over Pd black yields Ala-​Tyr-​Glu-​Pro-​Val-​Trp, part of the active site of the enzyme, triosephosphate isomerase

Peptides related to physalemin

C-​terminal peptide sequences of physalemin, X-​Phe-​Phe-​Phe-​Gly-​Leu-​Met-​NH2 (X = H, Asn, Pro-​Asn, Gln, Pro-​Gln)​, were prepd. by the solid phase method and had activities of 1.9, 7.0, 13.5, 3.7, and 2.7, resp., relative to 1 for bradykinin on guinea pig ileum

FIBRINOPEPTIDES. IV. : Synthesis of Human Fibrinopeptide a

Human fibrinopeptide A, a hexadecapeptide, released by the action of thrombin on fibrinogen during clotting of blood, has been synthesized by conventional methods. The synthetic peptide as well as some of the intermediates in the synthesis have been examined for anticoagulant activity. Though all of them were found to be active, the terminal carboxyl protected peptides are more potent inhibitors of clotting than the carboxyl free peptides

Fibrinopeptides. II. Synthesis of protected hexapeptide sequence (5-​10) of human fibrinopeptide-​A

The protected hexapeptide sequence (5-​10) of human fibrinopeptide-​A, BOC-​Glu(OBzl)​-​Gly-​Asp(OBzl)​-​Phe-​Leu-​Ala-​OMe (BOC = Me3CO2C, Bzl = PhCH2) (I)​, was synthesized using the mixed anhydride (MA)​, dicyclohexylcarbodiimide (DCC)​, 2,​4,​5-​trichlorophenyl ester (OCP) and pentachlorophenyl ester (OPCP) methods. The OCP method was applicable only after the tripeptide stage. The overall yield of I from the DCC, MA and OPCP methods was 23.2, 20.5 and 18.7​%, resp