A multi-lab experimental assessment reveals that replicability can be improved by using empirical estimates of genotype-by-lab interaction.

The utility of mouse and rat studies critically depends on their replicability in other laboratories. A widely advocated approach to improving replicability is through the rigorous control of predefined animal or experimental conditions, known as standardization. However, this approach limits the generalizability of the findings to only to the standardized conditions and is a potential cause rather than solution to what has been called a replicability crisis. Alternative strategies include estimating the heterogeneity of effects across laboratories, either through designs that vary testing conditions, or by direct statistical analysis of laboratory variation. We previously evaluated our statistical approach for estimating the interlaboratory replicability of a single laboratory discovery. Those results, however, were from a well-coordinated, multi-lab phenotyping study and did not extend to the more realistic setting in which laboratories are operating independently of each other. Here, we sought to test our statistical approach as a realistic prospective experiment, in mice, using 152 results from 5 independent published studies deposited in the Mouse Phenome Database (MPD). In independent replication experiments at 3 laboratories, we found that 53 of the results were replicable, so the other 99 were considered non-replicable. Of the 99 non-replicable results, 59 were statistically significant (at 0.05) in their original single-lab analysis, putting the probability that a single-lab statistical discovery was made even though it is non-replicable, at 59.6%. We then introduced the dimensionless "Genotype-by-Laboratory" (GxL) factor-the ratio between the standard deviations of the GxL interaction and the standard deviation within groups. Using the GxL factor reduced the number of single-lab statistical discoveries and alongside reduced the probability of a non-replicable result to be discovered in the single lab to 12.1%. Such reduction naturally leads to reduced power to make replicable discoveries, but this reduction was small (from 87% to 66%), indicating the small price paid for the large improvement in replicability. Tools and data needed for the above GxL adjustment are publicly available at the MPD and will become increasingly useful as the range of assays and testing conditions in this resource increases.

Mouse Phenome Database: towards a more FAIR-compliant and TRUST-worthy data repository and tool suite for phenotypes and genotypes.

The Mouse Phenome Database (MPD; https://phenome.jax.org; RRID:SCR_003212), supported by the US National Institutes of Health, is a Biomedical Data Repository listed in the Trans-NIH Biomedical Informatics Coordinating Committee registry. As an increasingly FAIR-compliant and TRUST-worthy data repository, MPD accepts phenotype and genotype data from mouse experiments and curates, organizes, integrates, archives, and distributes those data using community standards. Data are accompanied by rich metadata, including widely used ontologies and detailed protocols. Data are from all over the world and represent genetic, behavioral, morphological, and physiological disease-related characteristics in mice at baseline or those exposed to drugs or other treatments. MPD houses data from over 6000 strains and populations, representing many reproducible strain types and heterogenous populations such as the Diversity Outbred where each mouse is unique but can be genotyped throughout the genome. A suite of analysis tools is available to aggregate, visualize, and analyze these data within and across studies and populations in an increasingly traceable and reproducible manner. We have refined existing resources and developed new tools to continue to provide users with access to consistent, high-quality data that has translational relevance in a modernized infrastructure that enables interaction with a suite of bioinformatics analytic and data services.

Genes identified in rodent studies of alcohol intake are enriched for heritability of human substance use.

BACKGROUND: Rodent paradigms and human genome-wide association studies (GWAS) on drug use have the potential to provide biological insight into the pathophysiology of addiction. METHODS: Using GeneWeaver, we created rodent alcohol and nicotine gene-sets derived from 19 gene expression studies on alcohol and nicotine outcomes. We partitioned the SNP heritability of these gene-sets using four large human GWAS: (1) alcoholic drinks per week, (2) problematic alcohol use, (3) cigarettes per day, and (4) smoking cessation. We benchmarked our findings with curated human alcohol and nicotine addiction gene-sets and performed specificity analyses using other rodent gene-sets (e.g., locomotor behavior) and other human GWAS (e.g., height). RESULTS: The rodent alcohol gene-set was enriched for heritability of drinks per week, cigarettes per day, and smoking cessation, but not problematic alcohol use. However, the rodent nicotine gene-set was not significantly associated with any of these traits. Both rodent gene-sets showed enrichment for several non-substance-use GWAS, and the extent of this relationship tended to increase as a function of trait heritability. In general, larger gene-sets demonstrated more significant enrichment. Finally, when evaluating human traits with similar heritabilities, both rodent gene-sets showed greater enrichment for substance use traits. CONCLUSION: Our results suggest that rodent gene expression studies can help to identify genes that contribute to the heritability of some substance use traits in humans, yet there was less specificity than expected. We outline various limitations, interpretations, and considerations for future research.

Multi-omic and multi-species meta-analyses of nicotine consumption.

Cross-species translational approaches to human genomic analyses are lacking. The present study uses an integrative framework to investigate how genes associated with nicotine use in model organisms contribute to the genetic architecture of human tobacco consumption. First, we created a model organism geneset by collecting results from five animal models of nicotine exposure (RNA expression changes in brain) and then tested the relevance of these genes and flanking genetic variation using genetic data from human cigarettes per day (UK BioBank N = 123,844; all European Ancestry). We tested three hypotheses: (1) DNA variation in, or around, the 'model organism geneset' will contribute to the heritability to human tobacco consumption, (2) that the model organism genes will be enriched for genes associated with human tobacco consumption, and (3) that a polygenic score based off our model organism geneset will predict tobacco consumption in the AddHealth sample (N = 1667; all European Ancestry). Our results suggested that: (1) model organism genes accounted for ~5-36% of the observed SNP-heritability in human tobacco consumption (enrichment: 1.60-31.45), (2) model organism genes, but not negative control genes, were enriched for the gene-based associations (MAGMA, H-MAGMA, SMultiXcan) for human cigarettes per day, and (3) polygenic scores based on our model organism geneset predicted cigarettes per day in an independent sample. Altogether, these findings highlight the advantages of using multiple species evidence to isolate genetic factors to better understand the etiological complexity of tobacco and other nicotine consumption.

Mouse Phenome Database: a data repository and analysis suite for curated primary mouse phenotype data.

The Mouse Phenome Database (MPD; https://phenome.jax.org) is a widely accessed and highly functional data repository housing primary phenotype data for the laboratory mouse accessible via APIs and providing tools to analyze and visualize those data. Data come from investigators around the world and represent a broad scope of phenotyping endpoints and disease-related traits in naïve mice and those exposed to drugs, environmental agents or other treatments. MPD houses rigorously curated per-animal data with detailed protocols. Public ontologies and controlled vocabularies are used for annotation. In addition to phenotype tools, genetic analysis tools enable users to integrate and interpret genome-phenome relations across the database. Strain types and populations include inbred, recombinant inbred, F1 hybrid, transgenic, targeted mutants, chromosome substitution, Collaborative Cross, Diversity Outbred and other mapping populations. Our new analysis tools allow users to apply selected data in an integrated fashion to address problems in trait associations, reproducibility, polygenic syndrome model selection and multi-trait modeling. As we refine these tools and approaches, we will continue to provide users a means to identify consistent, quality studies that have high translational relevance.

Cisplatin-resistant triple-negative breast cancer subtypes: multiple mechanisms of resistance.

BACKGROUND: Understanding mechanisms underlying specific chemotherapeutic responses in subtypes of cancer may improve identification of treatment strategies most likely to benefit particular patients. For example, triple-negative breast cancer (TNBC) patients have variable response to the chemotherapeutic agent cisplatin. Understanding the basis of treatment response in cancer subtypes will lead to more informed decisions about selection of treatment strategies. METHODS: In this study we used an integrative functional genomics approach to investigate the molecular mechanisms underlying known cisplatin-response differences among subtypes of TNBC. To identify changes in gene expression that could explain mechanisms of resistance, we examined 102 evolutionarily conserved cisplatin-associated genes, evaluating their differential expression in the cisplatin-sensitive, basal-like 1 (BL1) and basal-like 2 (BL2) subtypes, and the two cisplatin-resistant, luminal androgen receptor (LAR) and mesenchymal (M) subtypes of TNBC. RESULTS: We found 20 genes that were differentially expressed in at least one subtype. Fifteen of the 20 genes are associated with cell death and are distributed among all TNBC subtypes. The less cisplatin-responsive LAR and M TNBC subtypes show different regulation of 13 genes compared to the more sensitive BL1 and BL2 subtypes. These 13 genes identify a variety of cisplatin-resistance mechanisms including increased transport and detoxification of cisplatin, and mis-regulation of the epithelial to mesenchymal transition. CONCLUSIONS: We identified gene signatures in resistant TNBC subtypes indicative of mechanisms of cisplatin. Our results indicate that response to cisplatin in TNBC has a complex foundation based on impact of treatment on distinct cellular pathways. We find that examination of expression data in the context of heterogeneous data such as drug-gene interactions leads to a better understanding of mechanisms at work in cancer therapy response.

Integration of heterogeneous functional genomics data in gerontology research to find genes and pathway underlying aging across species.

Understanding the biological mechanisms behind aging, lifespan and healthspan is becoming increasingly important as the proportion of the world's population over the age of 65 grows, along with the cost and complexity of their care. BigData oriented approaches and analysis methods enable current and future bio-gerontologists to synthesize, distill and interpret vast, heterogeneous data from functional genomics studies of aging. GeneWeaver is an analysis system for integration of data that allows investigators to store, search, and analyze immense amounts of data including user-submitted experimental data, data from primary publications, and data in other databases. Aging related genome-wide gene sets from primary publications were curated into this system in concert with data from other model-organism and aging-specific databases, and applied to several questions in genrontology using. For example, we identified Cd63 as a frequently represented gene among aging-related genome-wide results. To evaluate the role of Cd63 in aging, we performed RNAi knockdown of the C. elegans ortholog, tsp-7, demonstrating that this manipulation is capable of extending lifespan. The tools in GeneWeaver enable aging researchers to make new discoveries into the associations between the genes, normal biological processes, and diseases that affect aging, healthspan, and lifespan.

Mechanistic Differences in Neuropathic Pain Modalities Revealed by Correlating Behavior with Global Expression Profiling.

Chronic neuropathic pain is a major morbidity of neural injury, yet its mechanisms are incompletely understood. Hypersensitivity to previously non-noxious stimuli (allodynia) is a common symptom. Here, we demonstrate that the onset of cold hypersensitivity precedes tactile allodynia in a model of partial nerve injury, and this temporal divergence was associated with major differences in global gene expression in innervating dorsal root ganglia. Transcripts whose expression change correlates with the onset of cold allodynia were nociceptor related, whereas those correlating with tactile hypersensitivity were immune cell centric. Ablation of TrpV1 lineage nociceptors resulted in mice that did not acquire cold allodynia but developed normal tactile hypersensitivity, whereas depletion of macrophages or T cells reduced neuropathic tactile allodynia but not cold hypersensitivity. We conclude that neuropathic pain incorporates reactive processes of sensory neurons and immune cells, each leading to distinct forms of hypersensitivity, potentially allowing drug development targeted to each pain type.

Mouse Phenome Database: an integrative database and analysis suite for curated empirical phenotype data from laboratory mice.

The Mouse Phenome Database (MPD; https://phenome.jax.org) is a widely used resource that provides access to primary experimental trait data, genotypic variation, protocols and analysis tools for mouse genetic studies. Data are contributed by investigators worldwide and represent a broad scope of phenotyping endpoints and disease-related traits in naïve mice and those exposed to drugs, environmental agents or other treatments. MPD houses individual animal data with detailed, searchable protocols, and makes these data available to other resources via API. MPD provides rigorous curation of experimental data and supporting documentation using relevant ontologies and controlled vocabularies. Most data in MPD are from inbreds and other reproducible strains such that the data are cumulative over time and across laboratories. The resource has been expanded to include the QTL Archive and other primary phenotype data from mapping crosses as well as advanced high-diversity mouse populations including the Collaborative Cross and Diversity Outbred mice. Furthermore, MPD provides a means of assessing replicability and reproducibility across experimental conditions and protocols, benchmarking assays in users' own laboratories, identifying sensitized backgrounds for making new mouse models with genome editing technologies, analyzing trait co-inheritance, finding the common genetic basis for multiple traits and assessing sex differences and sex-by-genotype interactions.

Genetic regulation of Zfp30, CXCL1, and neutrophilic inflammation in murine lung.

Allergic asthma is a complex disease characterized in part by granulocytic inflammation of the airways. In addition to eosinophils, neutrophils (PMN) are also present, particularly in cases of severe asthma. We sought to identify the genetic determinants of neutrophilic inflammation in a mouse model of house dust mite (HDM)-induced asthma. We applied an HDM model of allergic asthma to the eight founder strains of the Collaborative Cross (CC) and 151 incipient lines of the CC (preCC). Lung lavage fluid was analyzed for PMN count and the concentration of CXCL1, a hallmark PMN chemokine. PMN and CXCL1 were strongly correlated in preCC mice. We used quantitative trait locus (QTL) mapping to identify three variants affecting PMN, one of which colocalized with a QTL for CXCL1 on chromosome (Chr) 7. We used lung eQTL data to implicate a variant in the gene Zfp30 in the CXCL1/PMN response. This genetic variant regulates both CXCL1 and PMN by altering Zfp30 expression, and we model the relationships between the QTL and these three endophenotypes. We show that Zfp30 is expressed in airway epithelia in the normal mouse lung and that altering Zfp30 expression in vitro affects CXCL1 responses to an immune stimulus. Our results provide strong evidence that Zfp30 is a novel regulator of neutrophilic airway inflammation.

Genetic variation in hippocampal microRNA expression differences in C57BL/6 J X DBA/2 J (BXD) recombinant inbred mouse strains.

BACKGROUND: miRNAs are short single-stranded non-coding RNAs involved in post-transcriptional gene regulation that play a major role in normal biological functions and diseases. Little is currently known about how expression of miRNAs is regulated. We surveyed variation in miRNA abundance in the hippocampus of mouse inbred strains, allowing us to take a genetic approach to the study of miRNA regulation, which is novel for miRNAs. The BXD recombinant inbred panel is a very well characterized genetic reference panel which allows quantitative trait locus (QTL) analysis of miRNA abundance and detection of correlates in a large store of brain and behavioural phenotypes. RESULTS: We found five suggestive trans QTLs for the regulation of miRNAs investigated. Further analysis of these QTLs revealed two genes, Tnik and Phf17, under the miR-212 regulatory QTLs, whose expression levels were significantly correlated with miR-212 expression. We found that miR-212 expression is correlated with cocaine-related behaviour, consistent with a reported role for this miRNA in the control of cocaine consumption. miR-31 is correlated with anxiety and alcohol related behaviours. KEGG pathway analysis of each miRNA's expression correlates revealed enrichment of pathways including MAP kinase, cancer, long-term potentiation, axonal guidance and WNT signalling. CONCLUSIONS: The BXD reference panel allowed us to establish genetic regulation and characterize biological function of specific miRNAs. QTL analysis enabled detection of genetic loci that regulate the expression of these miRNAs. eQTLs that regulate miRNA abundance are a new mechanism by which genetic variation influences brain and behaviour. Analysis of one of these QTLs revealed a gene, Tnik, which may regulate the expression of a miRNA, a molecular pathway and a behavioural phenotype. Evidence of genetic covariation of miR-212 abundance and cocaine related behaviours is strongly supported by previous functional studies, demonstrating the value of this approach for discovery of new functional roles and downstream processes regulated by miRNA.

Genotype-dependent effects of adolescent nicotine exposure on dopamine functional dynamics in the nucleus accumbens shell in male and female mice: a potential mechanism underlying the gateway effect of nicotine.

RATIONALE: The tendency to use cocaine is determined by genetic and environmental effects across the lifespan. One critical environmental effect is early drug exposure, which is both driven by and interacts with genetic background. The mesoaccumbens dopamine system, which is critically involved in the rewarding properties of drugs of abuse, undergoes significant development during adolescence, and thus may be at particular risk to repeated nicotine exposure during this period, thereby establishing vulnerability for subsequent adult psychostimulant use. OBJECTIVES: We tested the hypotheses that adolescent nicotine exposure results in attenuation of the enhancing effects of cocaine on medial forebrain bundle (MFB) electrical stimulation-evoked dopamine release in the nucleus accumbens shell (AcbSh) in adulthood and that this effect is significantly influenced by genotype. METHODS: Mice from the progenitor strains C57BL/6J and DBA/2J and those from the BXD20/TyJ and BXD86/RwwJ recombinant inbred lines were exposed to nicotine via osmotic minipumps from postnatal day (P) 28 to P56. When mice reached P70, dopamine functional dynamics in AcbSh was evaluated by means of in vivo fixed potential amperometry in combination with electrical stimulation of mesoaccumbens dopaminergic axons in the MFB. RESULTS: Adolescent exposure to nicotine in all strains dose-dependently reduced the ability of a fixed-dose challenge injection of cocaine (10 mg/kg, i.p.) to enhance MFB electrical stimulation-evoked dopamine release in AcbSh in adults. The magnitude of this effect was genotype-dependent. CONCLUSIONS: These results suggest a genotype-dependent mechanism by which nicotine exposure during adolescence causes persistent changes in the sensitivity to "hard" stimulants such as cocaine.

A genetic approach to the prediction of drug side effects: bleomycin induces concordant phenotypes in mice of the collaborative cross.

The antineoplastic drug bleomycin leads to the side effect of pulmonary fibrosis in both humans and mice. We challenged genetically diverse inbred lines of mice from the Collaborative Cross with bleomycin to determine the heritability of this phenotype. Sibling pairs of mice from 40 lines were treated with bleomycin. Lung disease was assessed by scoring lung pathology and by measuring soluble collagen levels in lavage fluid. Serum micro ribonucleic acids (miRNAs) were also measured. Inbred sibling pairs of animals demonstrated high coinheritance of the phenotypes of disease susceptibility or disease resistance. The plasma levels of one miRNA were clearly correlated in sibling mice. The results showed that, as in humans, the lines that comprise the Collaborative Cross exhibited wide genetic variation in response to this drug. This finding suggests that the genetically diverse Collaborative Cross animals may reveal drug effects that might be missed if a study were based on a conventional mouse strain.

The major locus for mouse adenovirus susceptibility maps to genes of the hematopoietic cell surface-expressed LY6 family.

Susceptibility to mouse adenovirus type 1 is associated with the major quantitative trait locus Msq1. Msq1 was originally mapped to a 13-Mb region of mouse chromosome (Chr) 15 in crosses between SJL/J and BALB/cJ inbred mice. We have now narrowed Msq1 to a 0.75-Mb interval from 74.68 to 75.43 Mb, defined by two anonymous markers, rs8259436 and D15Spn14, using data from 1396 backcross mice. The critical interval includes 14 Ly6 or Ly6-related genes, including Ly6a (encoding Sca-1/TAP), Ly6e (Sca-2/Tsa1), Ly6g (Gr-1), and gpihbp1 (GPI-anchored high-density lipoprotein-binding protein 1), as well as the gene encoding an aldosterone synthase (Cyp11b2). The Ly6 family members are attractive candidates for virus susceptibility genes because their products are GPI-anchored membrane proteins expressed on lymphoid and myeloid cells, with proposed functions in cell adhesion and cell signaling. To determine interstrain variation in susceptibility and produce additional resources for cloning Msq1, we assayed the susceptibility phenotype of four previously untested inbred mouse strains. Susceptibility of strain 129S6/SvEvTac was subsequently localized to the Ly6 complex region, using polymorphic genetic markers on Chr 15 in a population of 271 (129S6/SvEvTac x BALB/cJ)F(1) x BALB/cJ backcross mice. We identified a major 129S6/SvEvTac susceptibility allele, Msq1(129S6), on Chr 15 in the same region as Msq1(SJL). The results indicate that a major host factor in mouse adenovirus type 1 susceptibility is likely to be a member of the Ly6 gene family.

Extracting gene networks for low-dose radiation using graph theoretical algorithms.

Genes with common functions often exhibit correlated expression levels, which can be used to identify sets of interacting genes from microarray data. Microarrays typically measure expression across genomic space, creating a massive matrix of co-expression that must be mined to extract only the most relevant gene interactions. We describe a graph theoretical approach to extracting co-expressed sets of genes, based on the computation of cliques. Unlike the results of traditional clustering algorithms, cliques are not disjoint and allow genes to be assigned to multiple sets of interacting partners, consistent with biological reality. A graph is created by thresholding the correlation matrix to include only the correlations most likely to signify functional relationships. Cliques computed from the graph correspond to sets of genes for which significant edges are present between all members of the set, representing potential members of common or interacting pathways. Clique membership can be used to infer function about poorly annotated genes, based on the known functions of better-annotated genes with which they share clique membership (i.e., "guilt-by-association"). We illustrate our method by applying it to microarray data collected from the spleens of mice exposed to low-dose ionizing radiation. Differential analysis is used to identify sets of genes whose interactions are impacted by radiation exposure. The correlation graph is also queried independently of clique to extract edges that are impacted by radiation. We present several examples of multiple gene interactions that are altered by radiation exposure and thus represent potential molecular pathways that mediate the radiation response.

Complex genetics of interactions of alcohol and CNS function and behavior.

This work summarizes the proceedings of a symposium at the 2004 RSA Meeting in Vancouver, Canada. The organizers were R. W. Williams and D. B. Matthews; the Chair was M. F. Miles. The presentations were (1) WebQTL: A resource for analysis of gene expression variation and the genetic dissection of alcohol related phenotypes, by E. J. Chesler, (2) The marriage of microarray and qtl analyses: what's to gain, by J. K. Belknap, (3) Use of WebQTL to identify QTLs associated with footshock stress and ethanol related behaviors, by D. B. Matthews, (4) A high throughput strategy for the detection of quantitative trait genes, by R. J. Hitzemann, and (5) The use of gene arrays in conjunction with transgenic and selected animals to understand anxiety in alcoholism, by. B. Tabakoff.

Identification of quantitative trait loci for susceptibility to mouse adenovirus type 1.

Adult SJL/J mice are highly susceptible to mouse adenovirus type 1 (MAV-1) infections, whereas other inbred strains, including BALB/cJ, are resistant (K. R. Spindler, L. Fang, M. L. Moore, C. C. Brown, G. N. Hirsch, and A. K. Kajon, J. Virol. 75:12039-12046, 2001). Using congenic mouse strains, we showed that the H-2(s) haplotype of SJL/J mice is not associated with susceptibility to MAV-1. Susceptibility of MAV-1-infected (BALB/cJ x SJL/J)F(1) mice was intermediate between that of SJL/J mice and that of BALB/cJ mice, indicating that susceptibility is a genetically controlled quantitative trait. We mapped genetic loci involved in mouse susceptibility to MAV-1 by analysis of 192 backcross progeny in a genome scan with 65 simple sequence length polymorphic markers. A major quantitative trait locus (QTL) was detected on chromosome 15 (Chr 15) with a highly significant logarithm of odds score of 21. The locus on Chr 15 alone accounts for 40% of the total trait variance between susceptible and resistant strains. QTL modeling of the data indicated that there are a number of other QTLs with small effects that together with the major QTL on Chr 15 account for 54% of the trait variance. Identification of the major QTL is the first step in characterizing host genes involved in susceptibility to MAV-1.

Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'.

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

P2P-R expression is genetically coregulated with components of the translation machinery and with PUM2, a translational repressor that associates with the P2P-R mRNA.

P2P-R is a nuclear protein with potential functional roles in the control of gene expression and mitosis. The P2P-R protein also interacts with the p53 and Rb1 tumor suppressor proteins. To search for additional functional associations of P2P-R, we employed the WebQTL database that contains the results of cDNA microarray analysis on forebrain, cerebellum, and hematopoietic stem cell (HSC) specimens of multiple BXD recombinant inbred strains of mice. Using WebQTL, gene products were identified that show genetically based coexpression with P2P-R. Initial studies identified general groups of mRNAs that share common functional roles and high covariation in expression with P2P-R. These functional groups involved the regulation of transcription, nucleotide binding, translation control, and ion transport. The findings related to translational mechanisms were further evaluated. In HSCs, expression of P2P-R mRNA demonstrates an impressive expression correlation with a group of gene products associated with translation; high expression of P2P-R specifically was associated with decreased expression of 29 ribosomal protein mRNAs. In all three tissues that were screened using the WebQTL database, a strong positive expression covariance between P2P-R and the Pum2 gene product also was observed. PUM2 is a member of the highly conserved Puf family of RNA binding proteins that often function as gene-specific translation regulators. The ability of Puf proteins to repress translation is mediated by their binding to specific elements located in the 3' untranslated region (UTR) of their target mRNAs. To assess the functional significance of the strong genetic correlation in expression of P2P-R and PUM2, the 3' UTR of the P2P-R mRNA was analyzed and found to contain one perfect consensus and two near-perfect consensus PUM2 binding sequences. PUM2 pull-down methods combined with reverse transcription and RT-PCR confirmed that PUM2 does indeed bind P2P-R mRNA. These results suggest that P2P-R expression may be translationally regulated by PUM2 and that P2P-R may modulate translation by influencing ribosomal protein gene expression. This study represents the first description of a RNA target for mammalian Puf proteins and the first molecular confirmation of information obtained using the WebQTL database.

Acute progesterone can recruit sex-specific neurochemical mechanisms mediating swim stress-induced and kappa-opioid analgesia in mice.

There is a qualitative sex difference in the neurochemical mediation of stress-induced and kappa-opioid analgesia; these phenomena are dependent on N-methyl-d-aspartic acid (NMDA) receptors in males but not females. Progesterone modulation of this sex difference was examined in mice. Analgesia against thermal nociception was produced by forced cold water swim or by systemic administration of the kappa-opioid agonist, U50,488. As seen previously, the NMDA receptor antagonist MK-801 blocked both forms of analgesia in male but not female mice. Also as in previous studies, this sex difference was found to be dependent on ovarian hormones such that ovariectomy induced female mice to "switch" to the male-like, NMDAergic system. We now demonstrate that a single injection of progesterone (50 microg), systemically administered 30 min before analgesia assessment, is sufficient to restore female-specific mediation of analgesia (i.e., insensitivity to MK-801 blockade) in ovariectomized female mice. The rapidity of this neurochemical "switching" action of progesterone suggests mediation via cell surface receptors or the action of neuroactive steroid metabolites of progesterone.