Rabies virus infection is associated with variations in calbindin D-28K and calretinin mRNA expression levels in mouse brain tissue.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

Bacterial membrane vesicles and phages in blood after consumption of lacticaseibacillus rhamnosus JB-1.

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.

Whole-Genome Sequencing of Inbred Mouse Strains Selected for High and Low Open-Field Activity.

We conducted whole-genome sequencing of four inbred mouse strains initially selected for high (H1, H2) or low (L1, L2) open-field activity (OFA), and then examined strain distribution patterns for all DNA variants that differed between their BALB/cJ and C57BL/6J parental strains. Next, we assessed genome-wide sharing (3,678,826 variants) both between and within the High and Low Activity strains. Results suggested that about 10% of these DNA variants may be associated with OFA, and clearly demonstrated its polygenic nature. Finally, we conducted bioinformatic analyses of functional genomics data from mouse, rat, and human to refine previously identified quantitative trait loci (QTL) for anxiety-related measures. This combination of sequence analysis and genomic-data integration facilitated refinement of previously intractable QTL findings, and identified possible genes for functional follow-up studies.

Dose Fractionation During Puberty Is More Detrimental to Mammary Gland Development Than an Equivalent Acute Dose of Radiation Exposure.

PURPOSE: Computed tomographic (CT) scans in adolescents have increased dramatically in recent years. However, the effects of cumulative low-dose exposures on the development of radiation sensitive organs, such as the mammary gland, is unknown. The purpose of this work was to define the effects of dose rate on mammary organ formation during puberty, an especially sensitive window in mammary development. We used a fractionated low-dose x-ray exposure to mimic multiple higher dose CT scans, and we hypothesized that fractionated exposure would have less of an effect on the number of mammary gland defects compared with an acute exposure. METHODS AND MATERIALS: Female mice were subjected to fractionated low-dose x-ray exposure (10 cGy/d for 5 days), acute x-ray exposure (1 × 50 cGy), or sham exposure. As the wide genetic diversity in humans can play a role in a person's response to irradiation, 2 genetically diverse mouse strains differing in radiation sensitivity (BALB/c-sensitive; C57BL/6-resistant) were used to investigate the role of genetic background on the magnitude of the effect. RESULTS: Unexpectedly, our data reveal that multiple low-dose exposures produce greater immune and mammary defects for weeks after exposure compared with controls. The most pronounced defects being increased ductal branching in both strains and a greater percentage of terminal end buds in the BALB/c strain of mice exposed to fractionated radiation compared with sham. Radiation-induced defects near the terminal end bud were also increased in both strains. CONCLUSIONS: The findings suggest that fractionated low-dose exposures are potentially more damaging to organ development compared with an equivalent, single acute exposure and that genetic background is an important parameter modifying the severity of these effects.

Evaluation of skin irritation following weathered crude oil exposure in two mouse strains.

Petroleum crude oil spills are common and vary in size and scope. Spill response workers throughout the course of remediation are exposed to so-called weathered oil and are known to report diverse health effects, including contact dermatitis. A murine model of repeated exposure to weathered marine crude oil was employed utilizing two strains of mice, C57BL/6 and BALB/c, to investigate the pathology of this irritant and identify the principal hydrocarbon components deposited in skin. Histopathology demonstrated clear signs of irritation in oil-exposed skin from both mouse strains, characterized by prominent epidermal hyperplasia (acanthosis). BALB/c mice exposed to oil demonstrated more pronounced irritation compared with C57BL/6 mice, which was characterized by increased acanthosis as well as increased inflammatory cytokine/chemokine protein expression of IL-1ß, IL-6, CXCL10, CCL2, CCL3, CCL4, and CCL11. A gas chromatography/mass spectrometry method was developed for the identification and quantification of 42 aliphatic and EPA priority aromatic hydrocarbons from full thickness skin samples of C57BL/6 and BALB/c mice exposed to oil samples. Aromatic hydrocarbons were not detected in skin; however, aliphatic hydrocarbons in skin tended to accumulate with carbon numbers greater than C16. These preliminary data and observations suggest that weathered crude oil is a skin irritant and this may be related to specific hydrocarbon components, although immune phenotype appears to impact skin response as well.

Backcrossing to an appropriate genetic background improves the birth rate of carbohydrate sulfotransferase 14 gene-deleted mice.

Ehlers-Danlos syndromes (EDSs) are heterogeneous group of heritable connective tissue disorders characterized by joint and skin hyperextensibility as well as fragility of various organs. Recently, we described a new type of EDS, musculocontractual EDS (mcEDS-CHST14), caused by pathogenic variants of the carbohydrate sulfotransferase 14 (CHST14) gene mutation. B6;129S5-Chst14tm1Lex/Mmucd (B6;129-Chst14 KO) mice are expected to be an animal model of mcEDS-CHST14. However, >90% of B6;129-Chst14 KO homozygous (B6;129-Chst14-/-) mice show perinatal lethality. Therefore, improvement of the birth rate of Chst14-/- mice is needed to clarify the pathophysiology of mcEDS-CHST14 using this animal model. Some B6;129-Chst14-/- embryos had survived at embryonic day 18.5 in utero, suggesting that problems with delivery and/or childcare may cause perinatal lethality. However, in vitro fertilization and egg transfer did not improve the birth rate of the mice. A recent report showed that backcrossing to C57BL/6 strain induces perinatal death of all Chst14-/- mice, suggesting that genetic background influences the birthrate of these mice. In the present study, we performed backcrossing of B6;129-Chst14 KO mice to a BALB/c strain, an inbred strain that shows lower risks of litter loss than C57BL/6 strain. Upon backcrossing 1 to 12 times, the birth rate of Chst14-/- mice was improved with a birth rate of 6.12-18.64%. These results suggest that the genetic background influences the birth rate of Chst14-/- mice. BALB/c congenic Chst14-/- (BALB.Chst14-/-) mice may facilitate investigation of mcEDS-CHST14. Furthermore, backcrossing to an appropriate strain may contribute to optimizing animal experiments.

Methylation silencing of TGF-ß receptor type II is involved in malignant transformation of esophageal squamous cell carcinoma.

BACKGROUND: Although massive studies have been conducted to investigate the mechanisms of esophageal squamous cell carcinoma (ESCC) carcinogenesis, the understanding of molecular alterations during the malignant transformation of epithelial dysplasia is still lacking, especially regarding epigenetic changes. RESULTS: To better characterize the methylation changes during the malignant transformation of epithelial dysplasia, a whole-genome bisulfite sequencing analysis was performed on a series of tumor, dysplastic, and non-neoplastic epithelial tissue samples from esophageal squamous cell carcinoma (ESCC) patients. Promoter hypermethylation in TGF-ß receptor type II (TGFBR2), an important mediator of TGF-ß signaling, was identified. Further, we evaluated the methylation and expression of TGFBR2 in tumor samples through The Cancer Genome Atlas multiplatform data as well as immunohistochemistry. Moreover, treatment of ESCC cell lines with5-Aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, reactivated the expression of TGFBR2. The lentiviral mediating the overexpression of TGFBR2 inhibited the proliferation of ESCC cell line by inducing cell cycle G2/M arrest. Furthermore, the overexpression of TGFBR2 inhibited the tumor growth obviously in vivo. CONCLUSIONS: The characterization of methylation silencing of TGFBR2 in ESCC will enable us to further explore whether this epigenetic change could be considered as a predictor of malignant transformation in esophageal epithelial dysplasia and whether use of a TGFBR2 agonist may lead to a new therapeutic strategy in patients with ESCC.

Genetic regulation of antibody responsiveness to immunization in substrains of BALB/c mice.

Antibody-mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen-specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B-cell intrinsic defect in the regulation of class-switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class-switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

Scratching Responses to Epidermal Injury in C57BL/6, DBA/2, BALB/c, and CD1 Mice.

Whereas early investigations into ulcerative dermatitis (UD) focused on the possibility of a primary dermatopathology, several recent studies have advocated scratching behavior as a primary driver for UD. The aim of this study was to assess whether B6 mice exhibit excessive scratching under resting conditions or when provoked by epidermal barrier disruption. We hypothesized that B6 mice would exhibit more spontaneous scratching behavior and that B6 mice would be more pruritic after mild epidermal barrier injury compared with the other strains and stock tested. The behavior of the retired breeder female C57BL/6J, DBA/2J, BALB/cByJ, and Crl:CD1 mice was videotaped for 60 min. Behavior filming occurred at 17:15 and at 07:00 the next morning prior to (baseline) and after tape-stripping to initiate epidermal barrier disruption. Scratching duration was recorded as brief (less than 3 s) or prolonged (3 s or longer), on the basis of observations during a pilot study. In contrast to the hypothesis, B6 mice did not scratch significantly more frequently, have more long-duration scratching events, nor have a higher median scratching duration of prolonged scratching as compared with the other types of mice tested. In fact, B6 mice showed the lowest average scratching frequency and duration under both conditions. B6 mice demonstrated increased scratching behavior after epidermal barrier disruption, but the increased scratching did not surpass the rate or duration of scratching in the other types of mice tested. These findings do not support the idea that a strain-related tendency toward exaggerated scratching behavior under resting or epidermal barrier disruption conditions predisposes B6 mice to UD.

BALB/c-congenic ANP32B-deficient mice reveal a modifying locus that determines viability.

We previously found that deletion of the multifunctional factor ANP32B (a.k.a. SSP29, APRIL, PAL31, PHAPI2) resulted in a severe but strain-specific defect resulting in perinatal lethality. The difficulty in generating an adult cohort of ANP32B-deficient animals limited our ability to examine adult phenotypes, particularly cancer-related phenotypes. We bred the Anp32b-null allele into the BALB/c and FVB/N genetic background. The BALB/c, but not the FVB/N, background provided sufficient frequency of adult Anp32b-null (Anp32b(-/-)) animals. From these, we found no apparent oncogenic role for this protein in mammary tumorigenesis contrary to what was predicted based on human data. We also found runtism, pathologies in various organ systems, and an unusual clinical chemistry signature in the adult Anp32b(-/-) mice. Intriguingly, genome-wide single-nucleotide polymorphism analysis suggested that our colony retained an unlinked C57BL/6J locus at high frequency. Breeding this locus to homozygosity demonstrated that it had a strong effect on Anp32b(-/-) viability indicating that this locus contains a modifier gene of Anp32b with respect to development. This suggests a functionally important genetic interaction with one of a limited number of candidate genes, foremost among them being the variant histone gene H2afv. Using congenic breeding strategies, we have generated a viable ANP32B-deficient animal in a mostly pure background. We have used this animal to reliably exclude mouse ANP32B as an important oncogene in mammary tumorigenesis. Our further phenotyping strengthens the evidence that ANP32B is a widespread regulator of gene expression. These studies may also impact the choice of subsequent groups with respect to congenic breeding versus de novo zygote targeting strategies for background analyses in mouse genetics.

Behavioral characterization of a model of differential susceptibility to obesity induced by standard and personalized cafeteria diet feeding.

Despite the increase in obesity prevalence over the last decades, humans show large inter-individual variability for susceptibility to diet-induced obesity. Understanding the biological basis of this susceptibility could identify new therapeutic alternatives against obesity. We characterized behavioral changes associated with propensity to obesity induced by cafeteria (CAF) diet consumption in mice. We show that Balb/c mice fed a CAF diet display a large inter-individual variability in susceptibility to diet-induced obesity, such that based on changes in adiposity we can classify mice as obesity prone (OP) or obesity resistant (OR). Both OP and OR were hyperphagic relative to control-fed mice but caloric intake was similar between OP and OR mice. In contrast, OR had a larger increase in locomotor activity following CAF diet compared to OP mice. Obesity resistant and prone mice showed similar intake of sweet snacks, but OR ate more savory snacks than OP mice. Two bottle sucrose preference tests showed that OP decreased their sucrose preference compared to OR mice after CAF diet feeding. Finally, to test the robustness of the OR phenotype in response to further increases in caloric intake, we fed OR mice with a personalized CAF (CAF-P) diet based on individual snack preferences. When fed a CAF-P diet, OR increased their calorie intake compared to OP mice fed the standard CAF diet, but did not reach adiposity levels observed in OP mice. Together, our data show the contribution of hedonic intake, individual snack preference and physical activity to individual susceptibility to obesity in Balb/c mice fed a standard and personalized cafeteria-style diet.

Selective inbreeding does not increase gut microbiota similarity in BALB/c mice.

Inflammatory diseases in mouse models are under strong impact from the gut microbiota. Therefore increased interindividual gut microbiota similarity may be seen as a way to reduce group sizes in mouse experiments. The composition of the gut microbiota is to a high extent defined by genetics, and it is known that selecting siblings as mothers even in inbred colonies may increase the gut microbiota similarity among the mice with 3-4%. We therefore hypothesized that selective breeding of mice aiming at a high similarity in the gut microbiota would increase the interindividual similarity of the gut microbiota. BALB/cCrl mice were, however, found to have a mean heterozygosity of only 0.8% in their genome, and selection of breeders with a high similarity in the gut microbiota for three generations did not change the overall gut microbiota similarity, which was 66% in the P generation and 66%, 64% and 63% in the F1, F2 and F3 generations, respectively. Increased gut microbiota similarity in closely related mice in inbred mouse colonies is, therefore, more likely to be caused by other factors, such as imprinting or different intrauterine conditions, rather than by residual heterozygosity.

[Morphofunctional changes of BALB/c and C57BL/6 mice's immune system under chronic bacterial gram-negative endotoxicosis].

Chronic endotoxicosis was modeled by subcutaneous injection of the sepharose in complex with LPS. In these conditions we have studied morphofunctional changes of the immune system of BALB/c and C57Bl/6 mice, which are characterized by the different types of the immune response (Th2 type is predominant in BALB/c, Th1--in C57Bl/6). In the 1st-7th day t in the serum of BALB/c mice the endotoxin level increased in 21.3 times, in C57Bl/6--in 20.6 times. The endotoxin antibodies significantly decreased in 1th-7th days, on the 14th day it increased in the serum of both mice's strains. Morphofunctional changes of the immune system after chronic endotoxicosis were different in BALB/c and C57BI/6 mice. On the 1th day after injection of LPS and sepharose, in the thymus of C57Bl/6 mice the cortex layer was exhausted because of cell death, in the thymus of BALB/c mice II-III stages of accidental involution were developed. On the 7-14th day after injection of LPS and sepharose in the spleen of C57Bl/6 mice T- and B-zones were hyperplastic, however in spleen of BALB/c mice only T-zone were enlarged. After LPS and sepharose injection changes of cytokine production synthesized by KonA activated splenic cells were found out. In both strains the level of proinflammatory cytokines--TNFalpha and IL-1beta decreased, as well the Th1-cytokine IL-2. The production o fTh2-cytokine - IL-4, significantly decreased only in C57BI/6 mice. We suggest that damaging effect of LPS injection is determined by predominant Th2 or Th2 types of the immune response.

Experimental viral evolution to specific host MHC genotypes reveals fitness and virulence trade-offs in alternative MHC types.

The unprecedented genetic diversity found at vertebrate MHC (major histocompatibility complex) loci influences susceptibility to most infectious and autoimmune diseases. The evolutionary explanation for how these polymorphisms are maintained has been controversial. One leading explanation, antagonistic coevolution (also known as the Red Queen), postulates a never-ending molecular arms race where pathogens evolve to evade immune recognition by common MHC alleles, which in turn provides a selective advantage to hosts carrying rare MHC alleles. This cyclical process leads to negative frequency-dependent selection and promotes MHC diversity if two conditions are met: (i) pathogen adaptation must produce trade-offs that result in pathogen fitness being higher in familiar (i.e., host MHC genotype adapted to) vs. unfamiliar host MHC genotypes; and (ii) this adaptation must produce correlated patterns of virulence (i.e., disease severity). Here we test these fundamental assumptions using an experimental evolutionary approach (serial passage). We demonstrate rapid adaptation and virulence evolution of a mouse-specific retrovirus to its mammalian host across multiple MHC genotypes. Critically, this adaptive response results in trade-offs (i.e., antagonistic pleiotropy) between host MHC genotypes; both viral fitness and virulence is substantially higher in familiar versus unfamiliar MHC genotypes. These data are unique in experimentally confirming the requisite conditions of the antagonistic coevolution model of MHC evolution and providing quantification of fitness effects for pathogen and host. These data help explain the unprecedented diversity of MHC genes, including how disease-causing alleles are maintained.

Comparative analysis of the behavioral and biomolecular parameters of four mouse strains.

The use of mice as experimental models in pharmacological and biochemical research began over 100 years ago, during which time different mice strains with specific features were developed. Numerous studies demonstrate that the pharmacological efficacy of various compounds significantly varies among different animal strains, a factor which must be considered when analyzing experimental data. The Sabra strain, developed more than 35 years ago, is widely used for research in Israel but has an unclear origin and is not characterized as well as other strains. Comparative analyses of the molecular characteristics of Sabra and other strains should help to understand their characteristics and to enhance the validity of their experimental use. Thus, four mouse strains-outbred ICR and Sabra as well as inbred C57Bl/6J and Balb/c were compared. Animals' weight, blood corticosterone and hippocampal BDNF mRNA levels were measured, and animals' behavior was compared using the EPM, open field, FST, and hot plate tests. We found that although Sabra mice are bigger and heavier than other tested lines, this is not reflected in behavior or in biomolecular features, wherein Sabra mice lay within the diapason of other tested animals. Thus, behavioral tests of anxiety-like behavior and locomotor activity revealed that Sabra mice scored close to the mean of all tested lines. Analysis of blood corticosterone levels did not show significant differences among tested strains. We also found a correlation between general and locomotor activity of the tested strains and their hippocampal BDNF mRNA expression. In summary, we may conclude that Sabra mice have traits similar to the better known lines, and therefore they are good subjects for neuroscience research.

Comparative gene expression analysis in mouse models for identifying critical pathways in mammary gland development.

Functional development of the mammary gland is an important physiological process. Several studies have used gene expression profiling of mammary gland development to identify the key genes in the process, but few focused on the involved pathways. And there is a lack of concordance between those observed pathways. In this article, we applied a standardized microarray preprocessing to four independent studies, and then used gene set enrichment analysis (GSEA) to identify the key pathways. The result demonstrated an increased concordance between these expression profiling data sets. From the stage of puberty to pregnancy, we found 7 up-regulated and 6 down-regulated pathways in common. From the period of pregnancy to lactation, we found 7 up-regulated and 58 down-regulated pathways in common. And 10 up-regulated and 3 down-regulated pathways were found in common from lactation to the involution period. The main canonical pathways identified belong to immune system, cell communication, metabolism, and disease-related pathways. Pathway analysis is a more effective method than single gene analysis because the former one can able to detect genes weakly connected to the phenotype. As we applied GSEA to the study, our findings suggested a greater concordance in this physiological process. The critical pathways we find can provide some insight of functional development of the mammary gland and motivate other relative studies.

Identification of causal genes, networks, and transcriptional regulators of REM sleep and wake.

STUDY OBJECTIVE: Sleep-wake traits are well-known to be under substantial genetic control, but the specific genes and gene networks underlying primary sleep-wake traits have largely eluded identification using conventional approaches, especially in mammals. Thus, the aim of this study was to use systems genetics and statistical approaches to uncover the genetic networks underlying 2 primary sleep traits in the mouse: 24-h duration of REM sleep and wake. DESIGN: Genome-wide RNA expression data from 3 tissues (anterior cortex, hypothalamus, thalamus/midbrain) were used in conjunction with high-density genotyping to identify candidate causal genes and networks mediating the effects of 2 QTL regulating the 24-h duration of REM sleep and one regulating the 24-h duration of wake. SETTING: Basic sleep research laboratory. PATIENTS OR PARTICIPANTS: Male [C57BL/6J × (BALB/cByJ × C57BL/6J*) F1] N(2) mice (n = 283). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The genetic variation of a mouse N2 mapping cross was leveraged against sleep-state phenotypic variation as well as quantitative gene expression measurement in key brain regions using integrative genomics approaches to uncover multiple causal sleep-state regulatory genes, including several surprising novel candidates, which interact as components of networks that modulate REM sleep and wake. In particular, it was discovered that a core network module, consisting of 20 genes, involved in the regulation of REM sleep duration is conserved across the cortex, hypothalamus, and thalamus. A novel application of a formal causal inference test was also used to identify those genes directly regulating sleep via control of expression. CONCLUSION: Systems genetics approaches reveal novel candidate genes, complex networks and specific transcriptional regulators of REM sleep and wake duration in mammals.