Human KIT+ myeloid cells facilitate visceral metastasis by melanoma.

Metastasis of melanoma significantly worsens prognosis; thus, therapeutic interventions that prevent metastasis could improve patient outcomes. Here, we show using humanized mice that colonization of distant visceral organs with melanoma is dependent upon a human CD33+CD11b+CD117+ progenitor cell subset comprising \u3c4% of the human CD45+ leukocytes. Metastatic tumor-infiltrating CD33+ cells from patients and humanized (h)NSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNA-seq analysis identified a gene signature of a KIT/CD117-expressing CD33+ subset that correlated with decreased overall survival in a TCGA melanoma cohort. Thus, human CD33+CD11b+CD117+ myeloid cells represent a novel candidate biomarker as well as a therapeutic target for metastatic melanoma

Alu elements mediate MYB gene tandem duplication in human T-ALL

Recent studies have demonstrated that the MYB oncogene is frequently duplicated in human T cell acute lymphoblastic leukemia (T-ALL). We find that the human MYB locus is flanked by 257-bp Alu repeats and that the duplication is mediated somatically by homologous recombination between the flanking Alu elements on sister chromatids. Nested long-range PCR analysis indicated a low frequency of homologous recombination leading to MYB tandem duplication in the peripheral blood mononuclear cells of ∼50% of healthy individuals, none of whom had a MYB duplication in the germline. We conclude that Alu-mediated MYB tandem duplication occurs at low frequency during normal thymocyte development and is clonally selected during the molecular pathogenesis of human T-ALL

Alu elements mediate MYB gene tandem duplication in human T-ALL

Recent studies have demonstrated that the MYB oncogene is frequently duplicated in human T cell acute lymphoblastic leukemia (T-ALL). We find that the human MYB locus is flanked by 257-bp Alu repeats and that the duplication is mediated somatically by homologous recombination between the flanking Alu elements on sister chromatids. Nested long-range PCR analysis indicated a low frequency of homologous recombination leading to MYB tandem duplication in the peripheral blood mononuclear cells of ∼50% of healthy individuals, none of whom had a MYB duplication in the germline. We conclude that Alu-mediated MYB tandem duplication occurs at low frequency during normal thymocyte development and is clonally selected during the molecular pathogenesis of human T-ALL

PLS3 Missense Variants Affecting the Actin-Binding Domains Cause X-Linked Congenital Diaphragmatic Hernia and Body-Wall Defects

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G\u3eC (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder

The CMS Phase-1 pixel detector upgrade

The CMS detector at the CERN LHC features a silicon pixel detector as its innermost subdetector. The original CMS pixel detector has been replaced with an upgraded pixel system (CMS Phase-1 pixel detector) in the extended year-end technical stop of the LHC in 2016/2017. The upgraded CMS pixel detector is designed to cope with the higher instantaneous luminosities that have been achieved by the LHC after the upgrades to the accelerator during the first long shutdown in 2013–2014. Compared to the original pixel detector, the upgraded detector has a better tracking performance and lower mass with four barrel layers and three endcap disks on each side to provide hit coverage up to an absolute value of pseudorapidity of 2.5. This paper describes the design and construction of the CMS Phase-1 pixel detector as well as its performance from commissioning to early operation in collision data-taking.Peer reviewe

Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression

Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc(−/−) Ink4a/Arf(−/−) mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc(−/−) Ink4a/Arf(−/−) cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc(−/−) Ink4a/Arf(−/−) cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly, despite a marked increase in telomere reserve, cells derived from the ALT+ subcutaneous tumors were unable to generate lung metastases, indicating in vivo functional differences in these principal mechanisms of telomere maintenance. Together, these results are consistent with the model that although telomere dysfunction provokes chromosomal aberrations that initiate carcinogenesis, telomerase-mediated telomere maintenance enables such initiated cells to efficiently achieve a fully malignant endpoint, including metastasis

Rhbdf2 mutations increase its protein stability and drive EGFR hyperactivation through enhanced secretion of amphiregulin.

The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains. Proc Natl Acad Sci U S A 2014 May 27; 111(21):E2200-9

Loss of the high-affinity pentamidine transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [3H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1–/– line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4′,6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 μM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance

Generating Mouse Models Using CRISPR-Cas9-Mediated Genome Editing.

The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including mice. The CRISPR-Cas9 reagents can be delivered directly into the mouse zygote to derive a mutant animal carrying targeted genetic modifications. The major components of the system include the guide RNA, which provides target specificity, the Cas9 nuclease that creates the DNA double-strand break, and the donor oligonucleotide or plasmid carrying the intended mutation flanked by sequences homologous to the target site. Here we describe the general considerations and experimental protocols for creating genetically modified mice using the CRISPR-Cas9 system. © 2016 by John Wiley & Sons, Inc. Curr Protoc Mouse Biol 2016 Mar 1; 6(1):39-66