The major histocompatibility complex (MHC) plays a critical role in individual immune responses and susceptibility to various conditions, including autoimmune diseases and drug reactions. In dogs, the canine MHC (dog leukocyte antigen, DLA) polymorphism is key to understanding immune mechanisms, but technical challenges have impeded its comprehensive genetic analysis. This study addressed these issues by using a novel DLA genotyping method combining long-range PCR and PacBio single-molecule real-time sequencing to analyze the full-length DLA class I and II gene sequences in 83 beagle dogs from two different strains (TOYO and Marshall), which are commonly used as laboratory animals. As a result of genotyping using the full-length sequences, 9, 5, 2, 6, and 8 extended alleles were newly discovered for the DLA class I genes in DLA-88, DLA-12, DLA-88L, DLA-64, and DLA-79, respectively. For the DLA class II genes, 11, 18, 12, and 8 extended alleles were newly discovered in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. There were 25 haplotypes consisting of extended alleles, in contrast to only 10 haplotypes classified using only peptide binding site sequences. Furthermore, comparisons between the strains revealed differences in haplotype frequencies and genetic differentiation. The full-length analysis also provided preliminary insights into regulatory elements, such as promoter and CpG island polymorphisms in DLA-DQB1. The results of this research have important implications for the understanding of the relationship between DLA polymorphism at full length and individual immune responses in dogs.
The Cre-loxP system has been widely used in neuroscience for spatial and temporal control of gene expression. Although it is considered a powerful genetic tool, increasing evidence suggests that the Cre recombinase itself, without targeting loxP sites, may affect brain function and behavior. In this study, we assessed behavioral phenotypes in several Cre-driver rat lines, each expressing Cre under a different promoter (CAG, Pvalb, TH, Drd2, Tac1, or Thy1) to determine whether Cre expression influences behavioral outcomes. Behavioral testing included open field (for locomotor activity), hot plate (for nociceptive responses), prepulse inhibition (for sensorimotor gating), and contextual and cued fear conditioning (for associative learning). The Drd2-Cre rats exhibited significantly increased locomotor activity and movement speed compared to wild-type controls, suggesting heightened baseline activity. The CAG-Cre rats spent more time in the center area of the open field and showed reduced freezing during fear conditioning, suggesting changes in emotional regulation or learning. In contrast, no significant differences in the nociceptive responses or prepulse inhibition were observed in any of the Cre-driver lines. Following behavioral testing, we conducted structural MRI scans and found no obvious abnormalities in brain morphology across any of the Cre-driver lines, suggesting that the observed behavioral changes may reflect subtle neural alterations. These results suggested that Cre expression can produce promoter-dependent behavioral alterations, particularly in emotion or cognition. Therefore, behavioral validation of Cre-driver lines is recommended in neural circuit studies.
The cynomolgus monkey (Macaca fascicularis) is an important experimental animal; however, its menstrual patterns, lifespan, and age-related changes in anti-Müllerian hormone (AMH) levels remain poorly characterized. This study aimed to analyze these factors and evaluate the usefulness of Cynomolgus monkeys in ovarian function research. The age at menarche was examined in 21 cynomolgus monkeys, and the age at menopause and age at death were tracked in another 22 postmenopausal monkeys. In addition, AMH levels were analyzed in 74 cynomolgus monkeys aged 0 to 33 years to evaluate ovarian reserve throughout their lives. Results showed a mean age at menarche of 3.69 ± 2.51 years, menopause at 27.00 ± 2.50 years, and a mean age at death of 32.04 ± 5.33 years. AMH levels throughout life showed a weak negative correlation with age. These findings suggest that changes in ovarian reserve throughout the life span of cynomolgus monkeys are similar to those in humans. To our knowledge, this study is the first to analyze menstruation, lifespan, and lifelong AMH levels in cynomolgus monkeys. Ovarian function throughout life, including childhood and postmenopause, was similar to that in humans, suggesting cynomolgus monkeys may be a useful experimental model.
Xylene, an aromatic hydrocarbon, is widely used as a solvent in industries, such as printing and rubber and leather manufacturing. Despite the significant industrial benefits of xylene, there are increasing concerns about its environmental and health impacts. However, the effects of early exposure to xylene on the central nervous system are poorly understood. Here, using novel and unique approach, we investigated the neurobehavioral effects of xylene using postnatal lactation C57BL/6N male mice (Mus musculus) exposed to 0 (control) or 2 different doses (2 or 20 ppm) of xylene for 7 consecutive days at 22 h/d. The concentrations of xylene used corresponded respectively to 40 and 400 times the indoor air quality standard in Japan. We examined the effects of xylene on the murine central nervous system using a battery of behavioral tests, comprising the open field test, light-dark transition test, and contextual-cued fear conditioning test at 12 weeks of age. We found that mice exposed to xylene (2 and 20 ppm) presented reduced spatial-associative or tone-cued associative memory in the contextualcued fear conditioning test. In addition, immunohistochemical analysis revealed a decrease in doublecortin positive cells in the hippocampal dentate gyrus in the xylene exposure group compared with the control group. We confirmed that xylene exposure during postnatal lactation affects the formation of the neural circuit base and behavior in adulthood, suggesting that it is detrimental to postnatal brain development.
Macrophages can develop into pro-inflammatory M1-like macrophages and anti-inflammatory M2-like macrophages when stimulated by distinct internal environment. Dynamic changes of the two kinds of macrophages play key roles in atherosclerosis progression. The study aims to explore the role of ring finger protein 10 (RNF10) in regulating macrophage polarization during atherosclerosis. Mice with macrophage-specific depletion of RNF10 (RNF10Mac-KO/ApoE-/-) and control mice (RNF10fl/fl/ApoE-/-) mice were fed with high-fat diet to generate atherosclerotic lesion, from which peritoneal macrophages were isolated and transfected with RNF10-overexpressing vector. Murine macrophages, RAW264.7, were transfected with RNF10-overexpressing vector or RNF10 siRNA and stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The RNF10Mac-KO/ApoE-/- mice showed greater atherosclerotic lesions, more resident macrophages, higher expression of iNOS (M1-like macrophage marker), and lower expression of Arginase-1 (M2-like macrophage marker) than the RNF10fl/fl/ApoE-/- mice. RNF10 overexpression could reduce expressions of IL-1β, IL-6, and iNOS (M1 marker genes), increase expressions of IL-10 and Arg-1 (M2 marker genes) in the peritoneal macrophages isolated from RNF10Mac-KO/ApoE-/- mice. RNF10 overexpression reduced lipid accumulation in ox-LDL-induced foam cells, whereas RNF10 silencing yielded opposite results. Our data suggest that RNF10 is associated with M1-like macrophage suppression and M2-like increase, indicating RNF10 in macrophages has an anti-atherosclerotic role.
