RESUMO
Some of our breeding programs include the use of Prm1 male Homozygous mice which are naturally sterile. This removes the need to use vasectomized males to induce pseudopregnancy in female mice. These males can be kept for up to 9 months and are housed with a companion female. During the timed mating period the companion female is replaced with a new female. This procedure can occur at regular intervals causing a significant increase in cage activity; one of our objectives was to determine whether this was as a result of timed mating. We wanted to investigate the disruption caused to mice during the day of the swap and how long it would take for the cage activity to return to pre-replacement baseline levels. We hypothesized that this impact would be reflected as a significant increase in cage activity, which in itself may not be a result of a negative experience but the potential of repeated disruption to their activity pattern should be considered. We used a well-known home-cage monitoring system to assess changes to the activity pattern in cages when a companion female is replaced. Data from our initial study showed that in the 2-h period after the female is replaced there is a significant increase in cage activity compared to the same time frame on the previous day. In the subsequent study, where no cage change occurred, an increase in activity was also observed when females were replaced; this returned to baseline after approximately 4 h. Prolonged activity during the rest period of mice (over 2 h) could lead to them being fatigued during their active period; therefore, as a refinement we propose that timed matings be performed later in the day, at a time when the animals are active.
RESUMO
Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.