To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas 1 . Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.MicroRNAs (miRNAs) have emerged relatively recently as a new class of small, non-coding RNAs that regulate gene expression. Nascent primary miRNA transcripts (pri-miRNAs) are processed sequentially by two RNase III enzymes, Drosha and Dicer 2,3 , to yield mature miRNAs, ranging from 18 to 24 nucleotides (nt) in length. miRNAs are incorporated into the RNA interference (RNAi) effector complex, RISC, and target specific messenger RNAs Reprints and permissions information is available at npg.nature.com/reprintsandpermissionsCorrespondence and requests for materials should be addressed to G.J.H. (hannon@cshl.edu) or S.M.H. (hammond@med.unc.edu). * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Microarray data have been deposited in NCBI-GEO under accession numbers GSM45026-GSM45065 and GSE-2399.The authors declare no competing financial interests. -6 and miR-273 (refs 9, 10). Bantam stimulates cell growth and prevents apoptosis in Drosophila 11 , and miR-181 potentiates B-cell differentiation in mammals 12 . These findings, in combination with computational target predictions, are consistent with miRNAs regulating a broad spectrum of physiological and developmental processes. HHS Public AccessMicroarray-based expression studies have indicated specific alterations in human miRNA expression profiles that correlate with particular tumour phenotypes (J.M.T. and S.M.H., unpublished data). Among those that show altered expression, the mir-17-92 cistron is located at 13q31, a genomic locus that is amplified in cases of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, primary cutaneous B-cell lymphoma and several other tumour types 1,13 . There are only two annotated genes in the epicentre of this amplicon, c13orf25 and GPC5. Previous studies have shown that c13orf25 is the only one of the two genes for which increased expression correlates with the presence of the amplicon 1 . The...

The Collaborative Cross Consortium reports here on the development of a unique genetic resource population. The Collaborative Cross (CC) is a multiparental recombinant inbred panel derived from eight laboratory mouse inbred strains. Breeding of the CC lines was initiated at multiple international sites using mice from The Jackson Laboratory. Currently, this innovative project is breeding independent CC lines at the University of North Carolina (UNC), at Tel Aviv University (TAU), and at Geniad in Western Australia (GND). These institutions aim to make publicly available the completed CC lines and their genotypes and sequence information. We genotyped, and report here, results from 458 extant lines from UNC, TAU, and GND using a custom genotyping array with 7500 SNPs designed to be maximally informative in the CC and used a novel algorithm to infer inherited haplotypes directly from hybridization intensity patterns. We identified lines with breeding errors and cousin lines generated by splitting incipient lines into two or more cousin lines at early generations of inbreeding. We then characterized the genome architecture of 350 genetically independent CC lines. Results showed that founder haplotypes are inherited at the expected frequency, although we also consistently observed highly significant transmission ratio distortion at specific loci across all three populations. On chromosome 2, there is significant overrepresentation of WSB/EiJ alleles, and on chromosome X, there is a large deficit of CC lines with CAST/EiJ alleles. Linkage disequilibrium decays as expected and we saw no evidence of gametic disequilibrium in the CC population as a whole or in random subsets of the population. Gametic equilibrium in the CC population is in marked contrast to the gametic disequilibrium present in a large panel of classical inbred strains. Finally, we discuss access to the CC population and to the associated raw data describing the genetic structure of individual lines. Integration of rich phenotypic and genomic data over time and across a wide variety of fields will be vital to delivering on one of the key attributes of the CC, a common genetic reference platform for identifying causative variants and genetic networks determining traits in mammals.

Vigorous sperm motility, including the transition from progressive to hyperactivated motility that occurs in the female reproductive tract, is required for normal fertilization in mammals. We developed an automated, quantitative method that objectively classifies five distinct motility patterns of mouse sperm using Support Vector Machines (SVM), a common method in supervised machine learning. This multiclass SVM model is based on more than 2000 sperm tracks that were captured by computer-assisted sperm analysis (CASA) during in vitro capacitation and visually classified as progressive, intermediate, hyperactivated, slow, or weakly motile. Parameters associated with the classified tracks were incorporated into established SVM algorithms to generate a series of equations. These equations were integrated into a binary decision tree that sequentially sorts uncharacterized tracks into distinct categories. The first equation sorts CASA tracks into vigorous and nonvigorous categories. Additional equations classify vigorous tracks as progressive, intermediate, or hyperactivated and nonvigorous tracks as slow or weakly motile. Our CASAnova software uses these SVM equations to classify individual sperm motility patterns automatically. Comparisons of motility profiles from sperm incubated with and without bicarbonate confirmed the ability of the model to distinguish hyperactivated patterns of motility that develop during in vitro capacitation. The model accurately classifies motility profiles of sperm from a mutant mouse model with severe motility defects. Application of the model to sperm from multiple inbred strains reveals strain-dependent differences in sperm motility profiles. CASAnova provides a rapid and reproducible platform for quantitative comparisons of motility in large, heterogeneous populations of mouse sperm.

Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.

SUMMARY Evidence in vitro and in humans suggests that Mg2+ can alter systemic and renal vascular tone. However, the mechanism of these effects is not known. The role of vasodilator prostaglandin release and Ca 2+ flux in Mg 2+ -induced changes in blood pressure and renal blood flow was studied in 10 normal subjects maintained on a fixed 80-mEq Na + and K + diet. Magnesium sulfate infused at 200 mg/hr for 3 hours reduced systolic and diastolic blood pressure within 1 hour (from 119 ± 2 [SEM] to 109 ± 4 mm Hg systolic; from 74 ± 3 to 64 ± 4 mm Hg diastolic; p<0.02). This hypotensive response was seen in all subjects and persisted for 3 hours. The pulse rate did not change, but renal blood flow (p-aminohippurate clearance) increased (from 902 ± 78 to 1108 ± 130 ml/min/1.73 m 2 ; p<0.05). The Mg 2+ infusion produced a significant increase in the excretion of the stable prostaglandin I 2 (PGIj) metabolite 6-keto-PGF la (from 96 ± 12 to 154 ± 16 ng/g creatinine; p<0.01). In contrast, urinary PGEj was not altered (328 ± 75 vs 399 ± 145 ng/g creatinine; p>0.6).To evaluate the functional role of PGIj release, the cyclooxygenase inhibitors indomethacin (75 mg) or ibuprofen (600 mg) were given before the Mg* + infusion. Both cyclooxygenase blockers, given in doses that inhibited immunoreactive 6-keto-PGF, a release, completely prevented the Mg 2+ -induced decline in blood pressure and increased renal blood flow. In addition, pretreatment with the Ca 2+ channel antagonist nifedipine (20 mg Subjects and Methods Ten normal volunteers (7 men, 3 women), aged 21 to 44 years, were studied in the Clinical Research Center under informed consent after 4 days of equilibration on a constant 80-mEq Na + , 80-mEq K + diet. On 1 day, the subjects received an infusion of dextrose and water and a 3-hour urine sample was collected in parallel to serve as a control. On another day, the 379 Downloaded from http://ahajournals.org by on June 7, 2019