High throughput screening identified a substituted imidazole as a novel RANK pathway-selective osteoclastogenesis inhibitor.

Receptor activator of nuclear factor-kappaB (NF-kappaB) (RANK) plays a key role in the differentiation, activation, and survival of osteoclasts. Upon activation of RANK with RANK ligand (RANKL), osteoclast precursor cells differentiate into tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated osteoclasts. To identify compounds that block osteoclastogenesis, a cell-based assay was developed using RAW264.7 cells stably transfected with a TRAP promoter-dependent reporter gene as a surrogate readout for differentiation. Described herein is the strategy for high throughput screening and subsequent secondary biological assays for hit triage, which resulted in the identification of compound 1, a 4-nitroimidazole derivative, that specifically inhibited RANKL-induced TRAP gene and protein expression. Compound 1 did not affect the tumor necrosis factor-alpha- or lipopolysaccharide-induced TRAP-luciferase response, suggesting selective inhibition of the RANKL-induced pathway. Reverse transcription polymerase chain reaction analysis confirmed the inhibition of expression of osteoclast marker genes, such as TRAP, cathepsin K, and carbonic anhydrase type II. Compound 1 did not inhibit the RANKL-induced activation of a NF-kappaB reporter gene, or p38 kinase activity, suggesting a mechanism of action downstream of NF-kappaB. Together, these results suggest that we have identified a RANK pathway-specific inhibitor able to block the RANKL-induced osteoclast differentiation process. The hit identification strategy described here can be applied to other cell-based assays using an indirect surrogate readout to improve success rates.

Integrating identities.

Lesbians and gays who become step parents undergo a series of identity transitions. Previous models typically used to describe gay and lesbian identity formation lack attention to later life transitions. I argue that these models provide little understanding of the experiences of lesbians and gays who integrate their lesbian/gay identity with a step parent status. This transition is more complex than that experienced by either their partners (previously married biological parents) or by heterosexual step parents.

The identity transformation of biological parents in lesbian/gay stepfamilies.

This paper investigates the identity transformation of lesbian and gay biological parents in homosexual stepfamilies. I explore previous models typically used to describe gay and lesbian identity formation, arguing that these models provide little understanding of the experiences of those who have been previously married and have children. Lesbians and gays who leave heterosexual unions and form homosexual stepfamily units undergo a series of transformations. The results show that the transition is relatively positive and less internally stigmatizing and stressful than that experienced by younger, childless lesbians and gays.

Comparison of automated platelet counts and potential effect on transfusion decisions in cancer patients.

OBJECTIVES: To evaluate the accuracy of platelet counts from various hematology analyzers using a reference immunologic method. METHODS: We tested 403 samples with platelet counts less than 50 × 10(9)/L with the Advia (Siemens, Tarrytown, NY), Sysmex (Mundelein, IL), and Abbott (Santa Clara, CA) analyzers. RESULTS: All methods showed a positive bias, especially at less than 20 × 10(9)/L and less than 10 × 10(9)/L. Undertransfusion risk ranged from 9.1% to 43.3 % in the groups below 20 × 10(9)/L and below 10 × 10(9)/L, respectively. For patients with optical counts more than 10 × 10(9)/L and CD61 less than 10 × 10(9)/L, 64.5% were transfused within 24 hours of the reported count, while 35.5% were transfused in more than 24 hours, after a subsequent optical platelet count of 10 × 10(9)/L or less was reported. CONCLUSIONS: Although optical and impedance methods were shown to be falsely increased in severely thrombocytopenic samples, further studies are needed to determine if more accurate methods would be clinically useful.