Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-κB) signaling, as visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse¹.Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration^2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-κB luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-κB signaling within the LPS-injected gland⁴.These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.     

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.     

The proto-Nucleic Acid Builder: a software tool for constructing nucleic acid analogs

The helical structures of DNA and RNA were originally revealed by experimental data. Likewise, the development of programs for modeling these natural polymers was guided by known structures. These nucleic acid polymers represent only two members of a potentially vast class of polymers with similar structural features, but that differ from DNA and RNA in the backbone or nucleobases. Xeno nucleic acids (XNAs) incorporate alternative backbones that affect the conformational, chemical, and thermodynamic properties of XNAs. Given the vast chemical space of possible XNAs, computational modeling of alternative nucleic acids can accelerate the search for plausible nucleic acid analogs and guide their rational design. Additionally, a tool for the modeling of nucleic acids could help reveal what nucleic acid polymers may have existed before RNA in the early evolution of life. To aid the development of novel XNA polymers and the search for possible pre-RNA candidates, this article presents the proto-Nucleic Acid Builder (https://github.com/GT-NucleicAcids/pnab), an open-source program for modeling nucleic acid analogs with alternative backbones and nucleobases. The torsion-driven conformation search procedure implemented here predicts structures with good accuracy compared to experimental structures, and correctly demonstrates the correlation between the helical structure and the backbone conformation in DNA and RNA.     

Bone marrow hypoplasia associated with estrus in ferrets

Bone marrow hypoplasia was characterized in a group of female ferrets during prolonged estrus. All ferrets exhibited hematological changes characteristic of various degrees of bone marrow hypoplasia. Hematological findings included initial thrombocytosis and leukocytosis followed by thrombocytopenia, leukopenia and anemia. Platelet counts below 50,000/microliters were observed in 55% of the ferrets. Hemorrhagic anemia due to thrombocytopenia was the most common cause of death and the mortality rate was 40%. Histopathological findings included bone marrow hypoplasia affecting all cell lines and decreased splenic extramedullary hematopoiesis.     

Responsiveness to Retinoic Acid Changes during Chondrocyte Maturation

We previously showed that retinoic acid (RA) participates in the regulation of chondrocyte maturation during endochondral ossification, a process involving multiple developmental stages. To assess whether the responsiveness to RA treatment changes during chondrocyte maturation, immature chondrocytes were isolated from the caudal portion of Day 18-19 chick embryo sterna, a portion that remains cartilaginous through early postnatal life but ossifies with age. The immature cells were allowed to reach different stages of maturation by growth for different time in culture. Progression by the cells toward the mature phenotype during culture was confirmed by increases in average cell diameter, proteoglycan synthesis, and alkaline phosphatase (APase) activity. When developmentally immature passage 0 (PO) cultures were treated with RA (10-100 nM) for 72 h, the cells readily became fibroblastic, reduced drastically their proteoglycan synthesis, and failed to activate type X collagen gene expression. When older cultures (P1 and P2) were treated with RA, the cells acquired a characteristic epithelioid shape and increased their APase activity. Moreover, 5-10% of P1 cells and 20-25% of P2 cells activated type X collagen synthesis in response to RA. RA treatment markedly induced expression of the gene encoding the β isoform of retinoic acid receptor (RARβ) and also provoked a moderate 2.5-fold increase in RARα gene expression. A similar change in responsiveness to RA was observed during maturation in vivo. Chondrocytes were isolated from the cephalic portion of Day 10, 11, 13, and 16 chick embryo sterna, and were treated with different doses of RA (10-100 nM) for 72 h. The cells from the Day 10 sternum failed to activate type X collagen gene expression in response to RA. In contrast, with increasing age of the embryos, an increasing fraction of cells induced type X collagen gene expression in response to RA. We conclude that responsiveness to RA changes during the early stages of chondrocyte maturation and that maturation depends on interactions between exogenous retinoids and the endogenous developmental program of chondrocytes.