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Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1) diversity, to enable the signaling of multiple behaviors, 2) dynamic regulation, to indicate age and dominance, and 3) species-specificity. Recently, the major urinary proteins (Mups) have been shown to function themselves as geneticallyencoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues. Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species-specific function in mammals. [ABSTRACT FROM AUTHOR]

Mouse urine contains highly polymorphic major urinary proteins that have multiple functions in scent communication through their abilities to bind, transport and release hydrophobic volatile pheromones. The mouse genome encodes for about 20 of these proteins and are classified, based on amino acid sequence similarity and tissue expression patterns, as either central or peripheral major urinary proteins. Darcin is a male specific peripheral major urinary protein and is distinctive in its role in inherent female attraction. A comparison of the structure and biophysical properties of darcin with MUP11, which belongs to the central class, highlights similarity in the overall structure between the two proteins. The thermodynamic stability, however, differs between the two proteins, with darcin being much more stable. Furthermore, the affinity of a small pheromone mimetic is higher for darcin, although darcin is more discriminatory, being unable to bind bulkier ligands. These attributes are due to the hydrophobic ligand binding cavity of darcin being smaller, caused by the presence of larger amino acid side chains. Thus, the physical and chemical characteristics of the binding cavity, together with its extreme stability, are consistent with darcin being able to exert its function after release into the environment. [ABSTRACT FROM AUTHOR]

Male house mice produce large quantities of major urinary proteins (MUPs), which function to bind and transport volatile pheromones, though they may also function as scavengers that bind and excrete toxic compounds (‘toxic waste hypothesis’). In this study, we demonstrate the presence of an industrial chemical, 2,4-di-tert-butylphenol (DTBP), in the urine of wild-derived house mice (Mus musculus musculus). Addition of guanidine hydrochloride to male and female urine resulted in an increased release of DTBP. This increase was only observed in the high molecular weight fractions (HMWF; > 3 kDa) separated from male or female urine, suggesting that the increased release of DTBP was likely due to the denaturation of MUPs and the subsequent release of MUP-bound DTBP. Furthermore, when DTBP was added to a HMWF isolated from male urine, an increase in 2-sec-butyl-4,5-dihydrothiazole (SBT), the major ligand of MUPs and a male-specific pheromone, was observed, indicating that DTBP was bound to MUPs and displaced SBT. These results suggest that DTBP is a MUP ligand. Moreover, we found evidence for competitive ligand binding between DTBP and SBT, suggesting that males potentially face a tradeoff between eliminating toxic wastes versus transporting pheromones. Our findings support the hypothesis that MUPs bind and eliminate toxic wastes, which may provide the most important fitness benefits of excreting large quantities of these proteins. [ABSTRACT FROM AUTHOR]

Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the genomes of Mus musculus and Rattus norvegicus. We studied the evolutionary histories of the Mup and Esp genes and compared them with what is known about the Abp genes. Apparently gene conversion has played little if any role in the expansion of the mouse Class A and Class B Mup genes and pseudogenes, and the rat Mups. By contrast, we found evidence of extensive gene conversion in many Esp genes although not in all of them. Our studies of selection identified at least two amino acid sites in β-sheets as having evolved under positive selection in the mouse Class A and Class B MUPs and in rat MUPs. We show that selection may have acted on the ESPs by determining Ka/Ks for Exon 3 sequences with and without the converted sequence segment. While it appears that purifying selection acted on the ESP signal peptides, the secreted portions of the ESPs probably have undergone much more rapid evolution. When the inner gene converted fragment sequences were removed, eleven Esp paralogs were present in two or more pairs with Ka/Ks .1.0 and thus we propose that positive selection is detectable by this means in at least some mouse Esp paralogs. We compare and contrast the evolutionary histories of all three mouse pheromone gene families in light of their proposed functions in mouse communication. [ABSTRACT FROM AUTHOR]

Flavin-containing monooxygenase 5 (FMO5) is a member of the FMO family of proteins, best known for their roles in the detoxification of foreign chemicals and, more recently, in endogenous metabolism. We have previously shown that Fmo5-/- mice display an age-related lean phenotype, with much reduced weight gain from 20 weeks of age. The phenotype is characterized by decreased fat deposition, lower plasma concentrations of glucose, insulin and cholesterol, higher glucose tolerance and insulin sensitivity, and resistance to diet-induced obesity. In the present study we report the use of metabolomic and transcriptomic analyses of livers of Fmo5-/- and wild-type mice to identify factors underlying the lean phenotype of Fmo5-/- mice and gain insights into the function of FMO5. Metabolomics was performed by the Metabolon platform, utilising ultrahigh performance liquid chromatography-tandem mass spectroscopy. Transcriptomics was performed by RNA-Seq and results analysed by DESeq2. Disruption of the Fmo5 gene has wide-ranging effects on the abundance of metabolites and expression of genes in the liver. Metabolites whose concentration differed between Fmo5-/- and wild-type mice include several saturated and monounsaturated fatty acids, complex lipids, amino acids, one-carbon intermediates and ADP-ribose. Among the genes most significantly and/or highly differentially expressed are Apoa4, Cd36, Fitm1, Hspa5, Hyou1, Ide, Me1 and Mme. The results reveal that FMO5 is involved in upregulating the NRF2-mediated oxidative stress response, the unfolded protein response and response to hypoxia and cellular stress, indicating a role for the enzyme in adaptation to oxidative and metabolic stress. FMO5 also plays a role in stimulating a wide range of metabolic pathways and processes, particularly ones involved in lipid homeostasis, the uptake and metabolism of glucose, the generation of cytosolic NADPH, and in one-carbon metabolism. The results predict that FMO5 acts by stimulating the NRF2, XBP1, PPARA and PPARG regulatory pathways, while inhibiting STAT1 and IRF7 pathways. [ABSTRACT FROM AUTHOR]

Defective ribosome biogenesis (RiBi) underlies a group of clinically diverse human diseases collectively known as the ribosomopathies, core manifestations of which include cytopenias and developmental abnormalities that are believed to stem primarily from an inability to synthesize adequate numbers of ribosomes and concomitant activation of p53. The importance of a correctly functioning RiBi machinery for maintaining tissue homeostasis is illustrated by the observation that, despite having a paucity of certain cell types in early life, ribosomopathy patients have an increased risk for developing cancer later in life. This suggests that hypoproliferative states trigger adaptive responses that can, over time, become maladaptive and inadvertently drive unchecked hyperproliferation and predispose to cancer. Here we describe an experimentally induced ribosomopathy in the mouse and show that a normal level of hepatic ribosomal protein S6 (Rps6) is required for proper bile duct development and preservation of hepatocyte viability and that its insufficiency later promotes overgrowth and predisposes to liver cancer which is accelerated in the absence of the tumor-suppressor PTEN. We also show that the overexpression of c-Myc in the liver ameliorates, while expression of a mutant hyperstable form of p53 partially recapitulates specific aspects of the hepatopathies induced by Rps6 deletion. Surprisingly, co-deletion of p53 in the Rps6-deficient background fails to restore biliary development or significantly improve hepatic function. This study not only reveals a previously unappreciated dependence of the developing liver on adequate levels of Rps6 and exquisitely controlled p53 signaling, but suggests that the increased cancer risk in ribosomopathy patients may, in part, stem from an inability to preserve normal tissue homeostasis in the face of chronic injury and regeneration. Author summary: Ribosomopathies are a group of human diseases caused by mutations in genes that are required for ribosome biogenesis (RiBi), the process by which ribosomes, the molecular machines that synthesize all of the proteins in our bodies, are made. Here we show that mice that lack sufficient levels of one such gene, ribosomal protein S6 (Rps6) as the liver develops, fail to develop primary bile ducts resulting in disease reminiscent of North American Indian childhood cirrhosis (NAIC), a liver-specific ribosomopathy caused by mutations in a different RiBi gene, UTP4. Hepatocytes in Rps6-deficient livers also die resulting in small, abnormal livers that are forced to regenerate and predisposed to develop cancer. To determine the contribution of p53, a primary effector of the anti-proliferative response that is often triggered by dysfunctional RiBi to the liver disease caused by Rps6-deficiency, we find that forced activation of p53 inhibits bile duct development, but that the disease in Rps6-deficient livers is not solely p53-dependent. This study reveals a previously unappreciated vulnerability of the liver to defective RiBi or inappropriate activation of p53 and implicates both as possible drivers of a subset of congenital biliary or hepatic insufficiency syndromes for which there is currently no known etiology. [ABSTRACT FROM AUTHOR]

Mice respond to a cage change (CC) with altered activity, disrupted sleep and increased anxiety. A bi-weekly cage change is, therefore, preferred over a shorter CC interval and is currently the prevailing routine for Individually ventilated cages (IVCs). However, the build-up of ammonia (NH3) during this period is a potential threat to the animal health and the literature holds conflicting reports leaving this issue unresolved. We have therefor examined longitudinally in-cage activity, animal health and the build-up of ammonia across the cage floor with female and male C57BL/6 mice housed four per IVC changed every other week. We used a multicentre design with a standardised husbandry enabling us to tease-out features that replicated across sites from those that were site-specific. CC induce a marked increase in activity, especially during daytime (~50%) when the animals rest. A reduction in density from four to two mice did not alter this response. This burst was followed by a gradual decrease till the next cage change. Female but not male mice preferred to have the latrine in the front of the cage. Male mice allocate more of the activity to the latrine free part of the cage floor already the day after a CC. A behaviour that progressed through the CC cycle but was not impacted by the type of bedding used. Reducing housing density to two mice abolished this behaviour. Female mice used the entire cage floor the first week while during the second week activity in the latrine area decreased. Measurement of NH3 ppm across the cage floor revealed x3 higher values for the latrine area compared with the opposite area. NH3 ppm increases from 0–1 ppm to reach ≤25 ppm in the latrine free area and 50–100 ppm in the latrine area at the end of a cycle. As expected in-cage bacterial load covaried with in-cage NH3 ppm. Histopathological analysis revealed no changes to the upper airways covarying with recorded NH3 ppm or bacterial load. We conclude that housing of four (or equivalent biomass) C57BL/6J mice for 10 weeks under the described conditions does not cause any overt discomfort to the animals. [ABSTRACT FROM AUTHOR]

Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background. [ABSTRACT FROM AUTHOR]

The maned wolf (Chrysocyon brachyurus) is an induced ovulator. Though the mechanism of ovulation induction remains unknown, it is suspected to be urinary chemical signals excreted by males. This study assessed volatile organic compounds (VOCs) in weekly urine samples across 5 months from 13 maned wolves (6 intact males, 1 neutered male, 6 females) with the goal of identifying VOCs that are differentially expressed across sex, reproductive status, and pairing status. Solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were used to extract and separate VOCs that were identified via spectral matching with authentic standards, with spectral libraries, or with new software that further matches molecular fragment structures with mass spectral peaks. Two VOCs were present across all 317 urine samples: 2,5-dimethyl pyrazine and 2-methyl-6-(1-propenyl)-pyrazine. Fifteen VOCs differed significantly (Adj. P < 0.001 and |log2 fold change| >2.0) between intact males and females. Using partial least squares-discriminant analysis, the compounds with the highest importance to the sex classification were delta-decalactone, delta-dodecalactone, and bis(prenyl) sulfide. Sixty-two VOCs differed between intact males and the neutered male. Important classifier compounds were 3-ethyl 2,5-dimethyl pyrazine, 2-methyl-6-(1-propenyl)-pyrazine, and tetrahydro-2-isopentyl-5-propyl furan. Several VOCs established as important here have been implicated in reproductive communication in other mammals. This study is the most robust examination of differential expression in the maned wolf thus far and provides the most comprehensive analysis of maned wolf urinary VOCs to date, increasing the sample size substantially over previous chemical communication studies in this species. New data analysis software allowed for the identification of compounds in the hormone-producing mevalonate pathway which were previously unreported in maned wolf urine. Several putative semiochemicals were identified as good candidates for behavioral bioassays to determine their role in maned wolf reproduction, and specifically in ovulation induction. [ABSTRACT FROM AUTHOR]

The red fox is a highly adaptable mammal that has established itself world-wide in many different environments. Contributing to its success is a social structure based on chemical signalling between individuals. Urine scent marking behaviour has long been known in foxes, but there has not been a recent study of the chemical composition of fox urine. We have used solid-phase microextraction and gas chromatography-mass spectrometry to analyze the urinary volatiles in 15 free-ranging wild foxes (2 female) living in farmlands and bush in Victoria, Australia. Foxes here are routinely culled as feral pests, and the urine was collected by bladder puncture soon after death. Compounds were identified from their mass spectra and Kovats retention indices. There were 53 possible endogenous scent compounds, 10 plant-derived compounds and 5 anthropogenic xenobiotics. Among the plant chemicals were several aromatic apocarotenoids previously found in greater abundance in the fox tail gland. They reflect the dietary consumption of carotenoids, essential for optimal health. One third of all the endogenous volatiles were sulfur compounds, a highly odiferous group which included thiols, methylsulfides and polysulfides. Five of the sulfur compounds (3-isopentenyl thiol, 1- and 2-phenylethyl methyl sulfide, octanethiol and benzyl methyl sulfide) have only been found in foxes, and four others (isopentyl methyl sulfide, 3-isopentenyl methyl sulfide, and 1- and 2-phenylethane thiol) only in some canid, mink and skunk species. This indicates that they are not normal mammalian metabolites and have evolved to serve a specific role. This role is for defence in musteloids and most likely for chemical communication in canids. The total production of sulfur compounds varied greatly between foxes (median 1.2, range 0.4–32.3 μg 'acetophenone equivalents'/mg creatinine) as did the relative abundance of different chemical types. The urinary scent chemistry may represent a highly evolved system of semiochemicals for communication between foxes. [ABSTRACT FROM AUTHOR]

Ethologically relevant chemical senses and behavioral habits are likely to coadapt in response to selection. As olfaction is involved in intrinsically motivated behaviors in mice, we hypothesized that selective breeding for a voluntary behavior would enable us to identify novel roles of the chemosensory system. Voluntary wheel running (VWR) is an intrinsically motivated and naturally rewarding behavior, and even wild mice run on a wheel placed in nature. We have established 4 independent, artificially evolved mouse lines by selectively breeding individuals showing high VWR activity (High Runners; HRs), together with 4 non-selected Control lines, over 88 generations. We found that several sensory receptors in specific receptor clusters were differentially expressed between the vomeronasal organ (VNO) of HRs and Controls. Moreover, one of those clusters contains multiple single-nucleotide polymorphism loci for which the allele frequencies were significantly divergent between the HR and Control lines, i.e., loci that were affected by the selective breeding protocol. These results indicate that the VNO has become genetically differentiated between HR and Control lines during the selective breeding process. Although the role of the vomeronasal chemosensory receptors in VWR activity remains to be determined, the current results suggest that these vomeronasal chemosensory receptors are important quantitative trait loci for voluntary exercise in mice. We propose that olfaction may play an important role in motivation for voluntary exercise in mammals. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500–6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), the most malignant form of primary liver cancer, is the fourth most prevalent cause of cancer mortality globally. It was recently discovered that the dietary fermentable fiber, inulin, can reprogram the murine liver to favor HCC development in a gut microbiota-dependent manner. Determining the molecular pathways that are either over expressed or repressed during inulin-induced HCC would provide a platform of potential therapeutic targets. In the present study, we have combined analysis of the novel inulin-induced HCC murine model and human HCC samples to identify differentially expressed genes (DEGs) in hepatocarcinogenesis. Hepatic transcriptome profiling revealed that there were 674 DEGs in HCC mice compared to mice safeguarded from HCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered enrichment in ECM-receptor interaction, steroid hormone biosynthesis, PPAR signaling pathway, focal adhesion and protein digestion and absorption during inulin-induced HCC. Tandem mass tag based quantitative, multiplexed proteomic analysis delineated 57 differentially expressed proteins, where the over-expressed proteins were associated with cell adhesion molecules, valine, leucine and isoleucine degradation and ECM-receptor interaction. After obtaining the human orthologs of the mouse genes, we did a comparison analysis to level 3 RNA-seq data found in the Cancer Genome Atlas (TCGA) database, corresponding to human HCC (n = 361) and healthy liver (n = 50) samples. Out of the 549 up-regulated and 68 down-regulated human orthologs identified, 142 genes (137 significantly over-expressed and 5 significantly under-expressed) were associated with human HCC. Using univariate survival analysis, we found 27 over-expressed genes involved in cell-cell adhesion and cell division that were associated with poor HCC patient survival. Overall, the genetic and proteomics signatures highlight potential underlying mechanisms in inulin-induced HCC and support that this murine HCC model is human relevant. [ABSTRACT FROM AUTHOR]

For rodents, olfaction is essential for locating food, recognizing mates and competitors, avoiding predators, and navigating their environment. It is thought that rodents may have expanded olfactory receptor repertoires in order to specialize in olfactory behavior. Despite being the largest clade of mammals and depending on olfaction relatively little work has documented olfactory repertoires outside of conventional laboratory species. Here we report the olfactory receptor repertoire of the African giant pouched rat (Cricetomys ansorgei), a Muroid rodent distantly related to mice and rats. The African giant pouched rat is notable for its large cortex and olfactory bulbs relative to its body size compared to other sympatric rodents, which suggests anatomical elaboration of olfactory capabilities. We hypothesized that in addition to anatomical elaboration for olfaction, these pouched rats might also have an expanded olfactory receptor repertoire to enable their olfactory behavior. We examined the composition of the olfactory receptor repertoire to better understand how their sensory capabilities have evolved. We identified 1145 intact olfactory genes, and 260 additional pseudogenes within 301 subfamilies from the African giant pouched rat genome. This repertoire is similar to mice and rats in terms of size, pseudogene percentage and number of subfamilies. Analyses of olfactory receptor gene trees revealed that the pouched rat has 6 expansions in different subfamilies compared to mice, rats and squirrels. We identified 81 orthologous genes conserved among 4 rodent species and an additional 147 conserved genes within the Muroid rodents. The orthologous genes shared within Muroidea suggests that there may be a conserved Muroid-specific olfactory receptor repertoire. We also note that the description of this repertoire can serve as a complement to other studies of rodent olfaction, as the pouched rat is an outgroup within Muroidea. Thus, our data suggest that African giant pouched rats are capable of both natural and trained olfactory behaviors with a typical Muriod olfactory receptor repertoire. [ABSTRACT FROM AUTHOR]

Psychiatric disorders including depression and anxiety comprise a broad range of conditions with different symptoms. We have developed a mouse model of depression/anxiety in mice deficient in the St3gal4 gene. In this study, we performed a comparative analysis of urinary volatile organic compounds (VOCs) in St3gal4-deficient (St3gal4-KO) and wild-type mice using gas chromatography-mass spectrometry, and we screened 18 putative VOCs. Principal component analysis (PCA) based on these VOCs identified a major group of 11 VOCs, from which two groups were clarified by hierarchical clustering analysis. One group including six VOCs (pentanoic acid, 4-methyl-, ethyl ester; 3-heptanone, 6-methyl; benzaldehyde; 5,9-undecadien-2-ol, 6,10-dimethyl; and unknown compounds RI1291 and RI1237) was correlated with the startle response (r = 0.620), which is related to an unconscious defensive response. The other group including two VOCs (beta-farnesene and alpha-farnesene) comprised pheromones which increased in KO mice. Next, male mice underwent a social behavior test with female mice in the estrus stage, showing reduced access of KO male mice to female mice. Comparative analysis of urinary VOCs before and after encounters revealed that the six VOCs were not changed by these encounters. However, in WT mice, the two farnesenes increased after the encounters, reaching the level observed in KO mice, which was not altered following the encounter. Taken together, these results indicated that St3gal4 was involved in modulating urinary VOCs. Moreover, VOC clusters discovered by comparison of St3gal4-KO mice with WT mice were correlated with differential emotional behaviors. [ABSTRACT FROM AUTHOR]

For over a century, mice have been used to model human disease, leading to many fundamental discoveries about mammalian biology and the development of new therapies. Mouse genetics research has been further catalysed by a plethora of genomic resources developed in the last 20 years, including the genome sequence of C57BL/6J and more recently the first draft reference genomes for 16 additional laboratory strains. Collectively, the comparison of these genomes highlights the extreme diversity that exists at loci associated with the immune system, pathogen response, and key sensory functions, which form the foundation for dissecting phenotypic traits in vivo. We review the current status of the mouse genome across the diversity of the mouse lineage and discuss the value of mice to understanding human disease. Author summary: For decades, the laboratory mouse has been widely used to make fundamental discoveries about human biology, model human disease, and develop new treatments. The mouse reference genome is based on the C57BL/6J; however, researchers use a variety of strains to model human disease. Recent genome analysis has identified that the most highly variable regions of the mouse genome are enriched with genes relevant to disease and infection response. In this review, we discuss what is currently known about these regions, why they are important for human disease modelling, and what is known about their ancestral origins. [ABSTRACT FROM AUTHOR]

Di(2-ethylhexyl)phthalate (DEHP) interferes with sex hormones signaling pathways (SHP). C57BL/6J mice prenatally exposed to 300 mg/kg/day DEHP develop a testicular dysgenesis syndrome (TDS) at adulthood, but similarly-exposed FVB/N mice are not affected. Here we aim to understand the reasons behind this drastic difference that should depend on the genome of the strain. In both backgrounds, pregnant female mice received per os either DEHP or corn oil vehicle and the male filiations were examined. Computer-assisted sperm analysis showed a DEHP-induced decreased sperm count and velocities in C57BL/6J. Sperm RNA sequencing experiments resulted in the identification of the 62 most differentially expressed RNAs. These RNAs, mainly regulated by hormones, produced strain-specific transcriptional responses to prenatal exposure to DEHP; a pool of RNAs was increased in FVB, another pool of RNAs was decreased in C57BL/6J. In FVB/N, analysis of non-synonymous single nucleotide polymorphisms (SNP) impacting SHP identified rs387782768 and rs29315913 respectively associated with absence of the Forkhead Box A3 (Foxa3) RNA and increased expression of estrogen receptor 1 variant 4 (NM_001302533) RNA. Analysis of the role of SNPs modifying SHP binding sites in function of strain-specific responses to DEHP revealed a DEHP-resistance allele in FVB/N containing an additional FOXA1-3 binding site at rs30973633 and four DEHP-induced beta-defensins (Defb42, Defb30, Defb47 and Defb48). A DEHP-susceptibility allele in C57BL/6J contained five SNPs (rs28279710, rs32977910, rs46648903, rs46677594 and rs48287999) affecting SHP and six genes (Svs2, Svs3b, Svs4, Svs3a, Svs6 and Svs5) epigenetically silenced by DEHP. Finally, targeted experiments confirmed increased methylation in the Svs3ab promoter with decreased SEMG2 persisting across generations, providing a molecular explanation for the transgenerational sperm velocity decrease found in C57BL/6J after DEHP exposure. We conclude that the existence of SNP-dependent mechanisms in FVB/N inbred mice may confer resistance to transgenerational endocrine disruption. [ABSTRACT FROM AUTHOR]

The article focuses on how to understand the neurophysiology of phenotype and strength of the genetic approach is the unbiased nature of the screen. It mentions specific molecular stimulus identified the receptor and the afferent pathway, we are well on the way to creating a laboratory system, in vitro, that recapitulates the elements of the behavior. It also mentions chemical stimulus could be replaced by a recording electrode that detects an action potential from a neuron innervating muscle.

Ischemic stroke is one of the most common causes of death worldwide and is a major cause of acquired disability in adults. However, there is still a need for an effective drug for its treatment. Buyang Huanwu decoction (BHD), a traditional Chinese medicine (TCM) prescription, has long been used clinically to aid neurological recovery after stroke. To establish potential clinical indicators of BHD efficacy in stroke treatment and prognosis, we conducted a combined proteomic and metabolomic analysis of cerebrospinal fluid (CSF) samples in a mouse stroke model. CSF samples were obtained from male mice with acute ischemic stroke induced by middle cerebral ischemic/reperfusion (CI/R) injury, some of which were then treated with BHD. Label-free quantitative proteomics was conducted using nano-LC-MS/MS on an LTQ Orbitrap mass and metabolomic analysis was performed using nanoprobe NMR and UHPLC-QTOF-MS. The results showed that several proteins and metabolites were present at significantly different concentrations in the CSF samples from mice with CI/R alone and those treated with BHD. These belonged to pathways related to energy demand, inflammatory signaling, cytoskeletal regulation, Wnt signaling, and neuroprotection against neurodegenerative diseases. In conclusion, our in silico data suggest that BHD treatment is not only protective but can also ameliorate defects in pathways affected by neurological disorders. These data shed light on the mechanism whereby BHD may be effective in the treatment and prevention of stroke-related neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Pheromones, low molecular weight chemical entities that bind to pheromone carrier proteins, are chemical signals that play an important role in the communication system in animals. This has been rather fairly well-studied in the rodents. The preputial gland, a rich source of pheromones in many rodents, contains a low molecular mass protein (18–20 kDa) that acts as one such pheromone carrier. However, the presence of this protein in the notorious rodent pest Millardia meltada has not yet been proven. Therefore, we aimed at identifying this protein, and the pheromones that are bound to it, in this rodent so as to utilize the information in the control of this pest. Twenty volatile compounds were identified in the preputial gland using GC-MS. Total protein of the gland was fractioned by both one and two-dimensional electrophoresis when we identified a low molecular mass protein (19 kDa, pI-4.7). Adopting MALDI-TOF MS and LC-MS analyses, the protein was confirmed as α 2u-globulin. To identify the volatiles bound to this protein, we used column chromatography and GC-MS. We found that farnesol and 6-methyl-1-heptanol are the volatiles that would bind to the protein, which we propose to be putative pheromones. Immunohistochemical analysis confirmed localization of α 2u-globulin in the acinar cells of the preputial gland. Thus, we show that α 2u-globulin, a pheromone-carrier protein, is present in the preputial gland acinar cells of M. meltada and suggest farnesol and 6-methyl-1-heptanol to be the volatiles which would bind to it. The α 2u-globulin together with farnesol and 6-methyl-1-heptanol contribute to pheromonal communication of M. meltada. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AhR) is a highly conserved transcription factor that mediates a broad spectrum of species-, strain-, sex-, age-, tissue-, and cell-specific responses elicited by structurally diverse ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dose-dependent effects on liver-specific and sexually dimorphic gene expression were examined in male and female mice gavaged with TCDD every 4 days for 28 or 92 days. RNA-seq data revealed the coordinated repression of 181 genes predominately expressed in the liver including albumin (3.7-fold), α-fibrinogen (14.5-fold), and β-fibrinogen (17.4-fold) in males with corresponding AhR enrichment at 2 hr. Liver-specific genes exhibiting sexually dimorphic expression also demonstrated diminished divergence between sexes. For example, male-biased Gstp1 was repressed 3.0-fold in males and induced 4.5-fold in females, which were confirmed at the protein level. Disrupted regulation is consistent with impaired GHR-JAK2-STAT5 signaling and inhibition of female specific CUX2-mediated transcription as well as the repression of other key transcriptional regulators including Ghr, Stat5b, Bcl6, Hnf4a, Hnf6, Foxa1/2/3, and Zhx2. Attenuated liver-specific and sexually dimorphic gene expression was concurrent with the induction of fetal genes such as alpha-fetoprotein. The results suggest AhR activation causes the loss of liver-specific and sexually dimorphic gene expression producing a functionally “de-differentiated” hepatic phenotype. [ABSTRACT FROM AUTHOR]

In nanoflow liquid chromatography-matrix-assisted laser desorption/ionization tandem time-of-flight (nanoLC-MALDI-TOF/TOF) approaches, it is critical to directly apply small amounts of the sample elutes on the sample target using a nanoLC system due to its low flow rate of 200 ~ 300 nl/min. It is recommended to apply a sheath liquid containing a matrix with a several μL/min flow rate at the end of the nanoLC column to ensure a larger co-eluted droplet for more reproducible sample spotting and avoid the laborious task of post-manual matrix spotting. In this study, to achieve a better nanoLC-MALDI performance on sample spotting, we first compared α-Cyano-4-hydroxycinnamic acid (CHCA) solvent composition for efficiently concentrating nanoLC elutes on an anchor chip. The solvent composition of isopropanol (IPA): acetonitrile (ACN):acetone:0.1% Trifluoroacetic acid (TFA) (2:7:7:2) provided strong and homogeneous signals with higher peptide ion yields than the other solvent compositions. Then, nanoLC-MALDI-TOF/TOF was applied to study the impact of aflatoxin B1 on the liver proteome from diabetes mellitus type 1 mice. Aflatoxin B1 (AFB1), produced by Aspergillus flavus and Aspergillus parasiticus is a carcinogen and a known causative agent of liver cancer. To evaluate the effects of long-term exposure to AFB1 on type 1 diabetes mellitus (TIDM), the livers of T1DM control mice and mice treated with AFB1 were analyzed using isotope-coded protein labeling (ICPL)-based quantitative proteomics. Our results showed that gluconeogenesis, lipid, and oxidative phosphorylation mechanisms, normally elevated in T1DM, were disordered following AFB1 treatment. In addition, major urinary protein 1 (MUP1), an indicator of increased insulin sensitivity, was significantly decreased in the T1DM/AFB1 group and may have resulted in higher blood glucose levels compared to the T1DM group. These results indicate that T1DM patients should avoid the AFB1 intake, as they could lead to increased blood glucose levels and disorders of energy-producing mechanisms. [ABSTRACT FROM AUTHOR]

Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE) and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF), is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males—likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex. Courtship trials with females receiving different PMF isoform mixtures had variable effects on female mating receptivity, with only the most complex mixtures increasing receptivity, such that we believe that sufficient isoform diversity allows males to improve their reproductive success with any female in the mating population. The aim of this study was to test the effects of isoform variability on VNE neuron activation using the agmatine uptake assay. All isoform mixtures activated a similar number of neurons (>200% over background) except for a single purified PMF isoform (+17%). These data further support the hypothesis that PMF isoforms act synergistically in order to regulate female receptivity, and different putative mechanisms are discussed. [ABSTRACT FROM AUTHOR]

Intrasexual competition is an important element of natural selection in which the most attractive conspecific has a considerable reproductive advantage over the others. The conspecifics that are approached first often become the preferred mate partners, and could thus from a biological perspective have a reproductive advantage. This underlines the importance of the initial approach and raises the question of what induces this approach, or what makes a conspecific attractive. Identification of the sensory modalities crucial for the activation of approach is necessary for elucidating the central nervous processes involved in the activation of sexual motivation and eventually copulatory behavior. The initial approach to a potential mate depends on distant stimuli in the modalities of audition, olfaction, vision, and other undefined characteristics. This study investigated the role of the different modalities and the combination of these modalities in the sexual incentive value of a female rat. This study provides evidence that the presence of a single-sensory stimulus with one modality (olfaction, vision, or ‘others’, but not audition) is sufficient to attenuate the preference for a social contact with a male rat. However, a multisensory stimulus of multiple modalities is necessary to induce preference for the stimulus over social contact to a level of an intact receptive female. The initial approach behavior, therefore, seems to be induced by the combination of at least two modalities among which olfaction is crucial. This suggests that there is a cooperative function for the different modalities in the induction of approach behavior of a potential mate. [ABSTRACT FROM AUTHOR]

Woolly mammoths (Mammuthus primigenius) populated Siberia, Beringia, and North America during the Pleistocene and early Holocene. Recent breakthroughs in ancient DNA sequencing have allowed for complete genome sequencing for two specimens of woolly mammoths (Palkopoulou et al. 2015). One mammoth specimen is from a mainland population 45,000 years ago when mammoths were plentiful. The second, a 4300 yr old specimen, is derived from an isolated population on Wrangel island where mammoths subsisted with small effective population size more than 43-fold lower than previous populations. These extreme differences in effective population size offer a rare opportunity to test nearly neutral models of genome architecture evolution within a single species. Using these previously published mammoth sequences, we identify deletions, retrogenes, and non-functionalizing point mutations. In the Wrangel island mammoth, we identify a greater number of deletions, a larger proportion of deletions affecting gene sequences, a greater number of candidate retrogenes, and an increased number of premature stop codons. This accumulation of detrimental mutations is consistent with genomic meltdown in response to low effective population sizes in the dwindling mammoth population on Wrangel island. In addition, we observe high rates of loss of olfactory receptors and urinary proteins, either because these loci are non-essential or because they were favored by divergent selective pressures in island environments. Finally, at the locus of FOXQ1 we observe two independent loss-of-function mutations, which would confer a satin coat phenotype in this island woolly mammoth. [ABSTRACT FROM AUTHOR]

In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. [ABSTRACT FROM AUTHOR]

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process. [ABSTRACT FROM AUTHOR]

For many animals, chemosensation is essential for guiding social behavior. However, because multiple factors can modulate levels of individual chemical cues, deriving information about other individuals via natural chemical stimuli involves considerable challenges. How social information is extracted despite these sources of variability is poorly understood. The vomeronasal system provides an excellent opportunity to study this topic due to its role in detecting socially relevant traits. Here, we focus on two such traits: a female mouse’s strain and reproductive state. In particular, we measure stimulus-induced neuronal activity in the accessory olfactory bulb (AOB) in response to various dilutions of urine, vaginal secretions, and saliva, from estrus and non-estrus female mice from two different strains. We first show that all tested secretions provide information about a female’s receptivity and genotype. Next, we investigate how these traits can be decoded from neuronal activity despite multiple sources of variability. We show that individual neurons are limited in their capacity to allow trait classification across multiple sources of variability. However, simple linear classifiers sampling neuronal activity from small neuronal ensembles can provide a substantial improvement over that attained with individual units. Furthermore, we show that some traits are more efficiently detected than others, and that particular secretions may be optimized for conveying information about specific traits. Across all tested stimulus sources, discrimination between strains is more accurate than discrimination of receptivity, and detection of receptivity is more accurate with vaginal secretions than with urine. Our findings highlight the challenges of chemosensory processing of natural stimuli, and suggest that downstream readout stages decode multiple behaviorally relevant traits by sampling information from distinct but overlapping populations of AOB neurons. [ABSTRACT FROM AUTHOR]

Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones. [ABSTRACT FROM AUTHOR]

The maternal environment has been shown to influence female olfactory preferences through early chemosensory experience. However, little is known about the influence of the maternal environment on chemosignals. In this study, we used two inbred mouse strains, C57BL/6 (C57) and BALB/c (BALB), and explored whether adoption could alter male chemosignals and thus influence female olfactory preferences. In Experiment 1, C57 pups were placed with BALB dams. Adult BALB females then served as the subjects in binary choice tests between paired male urine odours (BALB vs. C57, BALB vs. adopted C57 and C57 vs. adopted C57). In Experiment 2, BALB pups were placed with C57 dams, and C57 females served as the subjects in binary choice tests between paired male urine odours (C57 vs. BALB, C57 vs. adopted BALB, and BALB vs. adopted BALB). In both experiments, we found that females preferred the urine of males from different genetic backgrounds, suggesting that female olfactory preferences may be driven by genetic compatibility. Cross-fostering had subtle effects on female olfactory preferences. Although the females showed no preference between the urine odours of adopted and non-adopted males of the other strain, the BALB females preferred the urine odour of BALB males to that of adopted C57 males, whereas the C57 females showed no preference between the urine odour of C57 and adopted BALB males. Using gas chromatography-mass spectrometry (GC-MS) and stepwise discriminant analysis, we found that the ratios of volatile chemicals from urine and preputial gland secretions were altered in the fostered male mice; these changes may have resulted in the behavioural changes observed in the females. Overall, the results suggest that female mice prefer urine odours from males with different genetic backgrounds; this preference may be driven by genetic compatibility. The early maternal environment influences the chemosignals of males and thus may influence the olfactory preferences of females. Our study provides additional evidence in support of genotype-dependent maternal influences on phenotypic variability in adulthood. [ABSTRACT FROM AUTHOR]

Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates. [ABSTRACT FROM AUTHOR]

The article enumerates the main narratives that have emerged in the past decade about biological mechanisms of behavioral change in rodents following toxoplasma infection. Topics cited include a narrative which notes that toxoplasma preferentially concentrates in certain brain regions which can explain host behavioral changes through local manipulation of neuronal signaling and the disruption of brain dopamine signaling as an important candidate in the nontropic model.

The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH). Mice fed a methionine and choline deficient diet (MCD) developed hepatic steatosis characterized by increased free fatty acid (FFA) and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx) is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress. [ABSTRACT FROM AUTHOR]

We have purified an abundant lipocalin from the seminal fluid of the rabbit, which shows significant similarity with the sub-class of pheromone carriers “urinary” and “salivary” and presents an N-terminal sequence identical with that of an odorant-binding protein (rabOBP3) expressed in the nasal tissue of the same species. This protein is synthesised in the prostate and found in the seminal fluid, but not in sperm cells. The same protein is also expressed in the nasal epithelium of both sexes, but is completely absent in female reproductive organs. It presents four cysteines, among which two are arranged to form a disulphide bridge, and is glycosylated. This is the first report of an OBP identified at the protein level in the seminal fluid of a vertebrate species. The protein purified from seminal fluid is bound to some organic chemicals whose structure is currently under investigation. We reasonably speculate that, like urinary and salivary proteins reported in other species of mammals, this lipocalin performs a dual role, as carrier of semiochemicals in the seminal fluid and as detector of chemical signals in the nose. [ABSTRACT FROM AUTHOR]

Diabetes is among the most common causes of end-stage renal disease, although its pathophysiology is incompletely understood. We performed next-generation sequencing-based transcriptome analysis of renal gene expression changes in the OVE26 murine model of diabetes (age 15 weeks), relative to non-diabetic control, in the presence and absence of short-term (seven-day) treatment with the angiotensin receptor blocker, losartan (n = 3–6 biological replicates per condition). We detected 1438 statistically significant changes in gene expression across conditions. Of the 638 genes dysregulated in diabetes relative to the non-diabetic state, >70% were downregulation events. Unbiased functional annotation of genes up- and down-regulated by diabetes strongly associated (p<1×10−8) with terms for oxidative stress and for endoplasmic reticulum stress/protein folding. Most of the individual gene products up- or down-regulated with diabetes were unaffected by losartan treatment; however, of the gene products dysregulated in diabetes and influenced by losartan treatment, the vast majority of changes were in the direction of amelioration rather than exacerbation of the diabetic dysregulation. This group of losartan-protected genes associated strongly with annotation terms for endoplasmic reticulum stress, heat shock proteins, and chaperone function, but not oxidative stress; therefore, the losartan-unaffected genes suggest avenues for additional therapeutic opportunity in diabetes. Interestingly, the gene product most highly upregulated by diabetes (>52-fold), encoded by the cationic amino acid transporter Slc7a12, and the gene product most highly downregulated by diabetes (>99%) – encoded by the “pseudogene” Gm6300 – are adjacent in the murine genome, are members of the SLC7 gene family, and are likely paralogous. Therefore, diabetes activates a near-total genetic switch between these two paralogs. Other individual-level changes in gene expression are potentially relevant to diabetic pathophysiology, and novel pathways are suggested. Genes unaffected by diabetes alone but exhibiting increased renal expression with losartan produced a signature consistent with malignant potential. [ABSTRACT FROM AUTHOR]

Male urinary lipocalin family proteins, practically odorant-binding proteins but also could be pheromones by themselves, in rodents act as a shuttle for chemosignal communication and facilitate delivery of the signals for access to congeners. However, presence of this protein in urine of female rodents has not yet been reported. Therefore, the present investigation was carried out to find if lipocalin family protein is present in the urine of female house rat and, if so, to find whether its expression differs between the phases in the estrous cycle. The rat urinary protein was separated in single dimensional gel electrophoresis. A 14.5 kDa lipocalin protein appeared in the urine prominently during the estrus and metestrus phases compared to proestrus and diestrus phases. The expression of this protein in the urine was very low in ovariectomized rats. MALDI-TOF/MS analysis affirmed the 14.5 kDa protein as a lipocalin family protein. Analysis adopting bio-informatics tools further proved the protein as a lipocalin family member. Thus, this study for the first time demonstrated the presence of a lipocalin family protein in the urine of a female rodent and it was highly expressed during estrus phase. This lipocalin protein in female rat urine may facilitate a chemosignal function independently of a pheromone or in association with a specific pheromone. [ABSTRACT FROM AUTHOR]

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. [ABSTRACT FROM AUTHOR]

General parameters of selection, such as the frequency and strength of positive selection in natural populations or the role of introgression, are still insufficiently understood. The house mouse (Mus musculus) is a particularly well-suited model system to approach such questions, since it has a defined history of splits into subspecies and populations and since extensive genome information is available. We have used high-density single-nucleotide polymorphism (SNP) typing arrays to assess genomic patterns of positive selection and introgression of alleles in two natural populations of each of the subspecies M. m. domesticus and M. m. musculus. Applying different statistical procedures, we find a large number of regions subject to apparent selective sweeps, indicating frequent positive selection on rare alleles or novel mutations. Genes in the regions include well-studied imprinted loci (e.g. Plagl1/Zac1), homologues of human genes involved in adaptations (e.g. alpha-amylase genes) or in genetic diseases (e.g. Huntingtin and Parkin). Haplotype matching between the two subspecies reveals a large number of haplotypes that show patterns of introgression from specific populations of the respective other subspecies, with at least 10% of the genome being affected by partial or full introgression. Using neutral simulations for comparison, we find that the size and the fraction of introgressed haplotypes are not compatible with a pure migration or incomplete lineage sorting model. Hence, it appears that introgressed haplotypes can rise in frequency due to positive selection and thus can contribute to the adaptive genomic landscape of natural populations. Our data support the notion that natural genomes are subject to complex adaptive processes, including the introgression of haplotypes from other differentiated populations or species at a larger scale than previously assumed for animals. This implies that some of the admixture found in inbred strains of mice may also have a natural origin. [ABSTRACT FROM AUTHOR]

NGAL/lipocalin-2 is a siderophore-binding protein that is highly expressed in several cancers. It is suggested to confer a proliferative advantage to cancer cells. Its expression has been correlated with aggressiveness of breast cancer as determined both in patients and in mouse breast cancer models. This was recently confirmed in two mouse models of spontaneous breast cancer in wild-type and lipocalin-2-deficient mice. We used a similar strategy using a different mouse strain. Lipocalin-2-deficient mice and mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) mice were crossed into the same FVB/N background. All mice developed tumors by week 8. The mice were sacrificed on week 13 and tissue was processed for biochemical and histological analysis. The total tumor volume and number of metastases were quantitated in 26 lipocalin-2-deficient mice and 34 wild-type controls. Lipocalin-2 expression in tumors of MMTV-PyMTpositive and wild-type mice was assessed by quantitative real-time PCR and by immunohistochemistry. The expression of the lipocalin-2 receptors 24p3R and megalin and of Mmp-9, transferrin receptor, and Bdh2 (a producer of a mammalian siderophore) were quantitated by real-time PCR. No significant difference was observed between wild-type and lipocalin-2- deficient mice. Lipocalin-2 was highly expressed in tumors from wild-type mice, but the expression did not correlate with tumor size. No effect of lipocalin-2 was observed with respect to time to tumor appearance, total tumor volume, or to the number of metastases. Histology and gelatinolytic activity of the mammary tumors did not differ between wild-type and lipocalin-2-deficient mice. We conclude that NGAL/lipocalin-2 does not invariably affect the aggressiveness of breast cancers as assessed in mouse models, thus questioning the role of lipocalin-2 in cancer development. [ABSTRACT FROM AUTHOR]

Scent marking can provide behavioral and physiological information including territory ownership and mate advertisement. It is unknown how mating status and pair cohabitation influence marking by males from different social systems. We compared the highly territorial and monogamous California mouse (Peromyscus californicus) to the less territorial and promiscuous white-footed mouse (P. leucopus). Single and mated males of both species were assigned to one of the following arenas lined with filter paper: control (unscented arena), male scented (previously scent-marked by a male conspecific), or females present (containing females in small cages). As expected, the territorial P. californicus scent marked and overmarked an unfamiliar male conspecific's scent marks more frequently than P. leucopus. Species differences in responses to novel females were also found based on mating status. The presence of unfamiliar females failed to induce changes in scent marking in pair bonded P. californicus even though virgin males increased marking behavior. Pair bonding appears to reduce male advertisement for novel females. This is in contrast to P. leucopus males that continue to advertise regardless of mating status. Our data suggest that communication through scent-marking can diverge significantly between species based on mating system and that there are physiological mechanisms that can inhibit responsiveness of males to female cues. [ABSTRACT FROM AUTHOR]

Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERa). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women. [ABSTRACT FROM AUTHOR]

Females in various species typically avoid males infected with parasites, while parasite-free males advertise their status through conspicuous phenotypic traits. This process selects for heritable resistance and reduces direct exposure of the female to parasites. Coevolving parasites are likely to attempt to circumvent this obstacle. In this paper, we demonstrate a case of parasitic manipulation of host mate choice. We report that Toxoplasma gondii, a sexually transmitted infection of brown rats, enhances sexual attractiveness of infected males. Thus under some evolutionary niches, parasites can indeed manipulate host sexual signaling to their own advantage. [ABSTRACT FROM AUTHOR]

Although behavioral and endocrine consequences of acute exposure to stressors have been extensively studied, little is known about how simultaneous exposure to two different stressors interacts to induce short- and long-term effects. In the present experiment we studied this interaction in adult male rats exposed to cat fur odor (impregnated cloth) or immobilization on boards either separately or simultaneously. We reasoned that exposure to the odor of a potential predator while immobilized, may potentiate its negative consequences as compared to exposure to only one of the stressors. Exposure to cat odor elicited the expected reduction of activity and avoidance of the area where the impregnated cloth was located. The endocrine response (plasma levels of ACTH and corticosterone, as a measure of the hypothalamicpituitary- adrenal axis, HPA) was markedly greater after immobilization than after cat fur odor and no additive effects were found by simultaneous exposure to both stressors. Cat odor, but not immobilization, increased anxiety-like behavior as evaluated in the elevated plus-maze 7 days after the stressors, with no evidence of enhanced HPA activation. In addition, cat odor exposure resulted in long-lasting (8 days later) fear conditioning to the box containing a clean cloth, which was reflected by hypoactivity, avoidance of the cloth area and enhanced HPA activation. All these effects were similarly observed in rats exposed simultaneously to cat odor and immobilization. In rats only exposed to immobilization, only some weak behavioral signs of fear conditioning were found, but HPA activation in response to the context paired to immobilization was enhanced to the same extent as in cat odor-exposed animals, supporting a certain degree of endocrine conditioning. The present results did not reveal important behavioral interactions between the two stressors when animals experienced both simultaneously, whereas some interactions were found regarding HPA activation. Theoretical implications are discussed. [ABSTRACT FROM AUTHOR]

We performed proteomics studies of salivas from the genome mouse (C57BL/6 strain) and the genome rat (BN/SsNHsd/ Mcwi strain). Our goal was to identify salivary proteins with one or more of three characteristics that may indicate that they have been involved in adaptation: 1) rapid expansion of their gene families; 2) footprints of positive selection; and/or 3) sexlimited expression. The results of our proteomics studies allow direct comparison of the proteins expressed and their levels between the sexes of the two rodent species. Twelve members of the Mus musculus species-specific kallikrein subfamily Klk1b showed sex-limited expression in the mouse saliva proteomes. By contrast, we did not find any of the Rattus norvegicus species-specific kallikrein subfamily Klk1c proteins in male or female genome rat, nor transcripts in their submandibular glands. On the other hand, we detected expression of this family as transcripts in the submandibular glands of both sexes of Sprague-Dawley rats. Using the CODEML program in the PAML package, we demonstrate that the two rodent kallikrein subfamilies have apparently evolved rapidly under the influence of positive selection that continually remodeled the amino acid sites on the same face in the members of the subfamilies. Thus, although their kallikrein subfamily expansions were independent, this evolutionary pattern has occurred in parallel in the two rodent species, suggesting a form of convergent evolution at the molecular level. On the basis of this new data, we suggest that the previous speculative function of the species-specific rodent kallikreins as important solely in wound healing in males be investigated further. In addition to or instead of that function, we propose that their sex-limited expression, coupled with their rapid evolution may be clues to an as-yet-undetermined interaction between the sexes. [ABSTRACT FROM AUTHOR]

Male odors can influence a female's reproductive physiology. In the mouse, the odor of male urine results in an early onset of female puberty. Several volatile and protein pheromones have previously been reported to each account for this bioactivity. Here we bioassay inbred BALB/cJ females to study pheromone-accelerated uterine growth, a developmental hallmark of puberty. We evaluate the response of wild-type and mutant mice lacking a specialized sensory transduction channel, TrpC2, and find TrpC2 function to be necessary for pheromone-mediated uterine growth. We analyze the relative effectiveness of pheromones previously identified to accelerate puberty through direct bioassay and find none to significantly accelerate uterine growth in BALB/cJ females. Complementary to this analysis, we have devised a strategy of partial purification of the uterine growth bioactivity from male urine and applied it to purify bioactivity from three different laboratory strains. The biochemical characteristics of the active fraction of all three strains are inconsistent with that of previously known pheromones. When directly analyzed, we are unable to detect previously known pheromones in urine fractions that generate uterine growth. Our analysis indicates that pheromones emitted by males to advance female puberty remain to be identified. [ABSTRACT FROM AUTHOR]

Background: Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice. Methodology/Principal Findings: Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1-2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months. Conclusions: E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are extensively involved in diverse biological processes. However, very little is known about the role of miRNAs in mediating the action of thyroid hormones (TH). Appropriate TH levels are known to be critically important for development, differentiation and maintenance of metabolic balance in mammals. We induced transient hypothyroidism in juvenile mice by short-term exposure to methimazole and perchlorate from post natal day (PND) 12 to 15. The expression of miRNAs in the liver was analyzed using Taqman Low Density Arrays (containing up to 600 rodent miRNAs). We found the expression of 40 miRNAs was significantly altered in the livers of hypothyroid mice compared to euthyroid controls. Among the miRNAs, miRs-1, 206, 133a and 133b exhibited a massive increase in expression (50- to 500-fold). The regulation of TH on the expression of miRs-1, 206, 133a and 133b was confirmed in various mouse models including: chronic hypothyroid, short-term hyperthyroid and short-term hypothyroid followed by TH supplementation. TH regulation of these miRNAs was also confirmed in mouse hepatocyte AML 12 cells. The expression of precursors of miRs-1, 206, 133a and 133b were examined in AML 12 cells and shown to decrease after TH treatment, only pre-mir-206 and pre-mir-133b reached statistical significance. To identify the targets of these miRNAs, DNA microarrays were used to examine hepatic mRNA levels in the short-term hypothyroid mouse model relative to controls. We found transcripts from 92 known genes were significantly altered in these hypothyroid mice. Web-based target predication software (TargetScan and Microcosm) identified 14 of these transcripts as targets of miRs-1, 206, 133a and 133b. The vast majority of these mRNA targets were significantly downregulated in hypothyroid mice, corresponding with the up-regulation of miRs-1, 206, 133a and 133b in hypothyroid mouse liver. To further investigate target genes, miR-206 was over-expressed in AML 12 cells. TH treatment of cells over-expressing miR-206 resulted in decreased miR-206 expression, and a significant increase in two predicted target genes, Mup1 and Gpd2. The results suggest that TH regulation of these genes may occur secondarily via miR-206. These studies provide new insight into the role of miRNAs in mediating TH regulation of gene expression. [ABSTRACT FROM AUTHOR]

The article offers information on a study conducted on the transposition and intermingling of Gαi2 and Gαo afferences into single vomeronasal glomeruli in the lesser Tenrec Echinops telfairi. It states that the proteins are arranged in rostral and caudal territories, and the density of mitral/ tufted cell layer is not homogeneous across the rostro-caudal axis of accessory olfactory bulb (AOB). It mentions that all mammals with two-pathway VNS showed clear-cut segregation of Gαi2 and Gαo positive glomeruli.

Background: Experiments under controlled laboratory conditions can produce decisive evidence for testing biological hypotheses, provided they are representative of the more complex natural conditions. However, whether this requirement is fulfilled is seldom tested explicitly. Here we provide a lab/field comparison to investigate the identity of an egg-marking signal of ant queens. Our study was based on ant workers resolving conflict over male production by destroying each other's eggs, but leaving queen eggs unharmed. For this, the workers need a proximate cue to discriminate between the two egg types. Earlier correlative evidence indicated that, in the ant Pachycondyla inversa, the hydrocarbon 3,11- dimethylheptacosane (3,11-diMeC27) is more abundant on the surface of queen-laid eggs. Methodology: We first tested the hypothesis that 3,11-diMeC27 functions as a queen egg-marking pheromone using laboratory-maintained colonies. We treated worker-laid eggs with synthetic 3,11-diMeC27 and found that they were significantly more accepted than sham-treated worker-laid eggs. However, we repeated the experiment with freshly collected field colonies and observed no effect of treating worker-laid eggs with 3,11-diMeC27, showing that this compound by itself is not the natural queen egg-marking pheromone. We subsequently investigated the overall differences of entire chemical profiles of eggs, and found that queen-laid eggs in field colonies are more distinct from worker-laid eggs than in lab colonies, have more variation in profiles, and have an excess of longer-chain hydrocarbons. Conclusions: Our results suggest that queen egg-marking signals are significantly affected by transfer to the laboratory, and that this change is possibly connected to reduced queen fertility as predicted by honest signaling theory. This change is reflected in the worker egg policing response under field and laboratory conditions. [ABSTRACT FROM AUTHOR]

Individual mice have a unique odor, or odortype, that facilitates individual recognition. Odortypes, like other phenotypes, can be influenced by genetic and environmental variation. The genetic influence derives in part from genes of the major histocompatibility complex (MHC). A major environmental influence is diet, which could obscure the genetic contribution to odortype. Because odortype stability is a prerequisite for individual recognition under normal behavioral conditions, we investigated whether MHC-determined urinary odortypes of inbred mice can be identified in the face of large diet-induced variation. Mice trained to discriminate urines from panels of mice that differed both in diet and MHC type found the diet odor more salient in generalization trials. Nevertheless, when mice were trained to discriminate mice with only MHC differences (but on the same diet), they recognized the MHC difference when tested with urines from mice on a different diet. This indicates that MHC odor profiles remain despite large dietary variation. Chemical analyses of urinary volatile organic compounds (VOCs) extracted by solid phase microextraction (SPME) and analyzed by gas chromatography/mass spectrometry (GC/MS) are consistent with this inference. Although diet influenced VOC variation more than MHC, with algorithmic training (supervised classification) MHC types could be accurately discriminated across different diets. Thus, although there are clear diet effects on urinary volatile profiles, they do not obscure MHC effects. [ABSTRACT FROM AUTHOR]