Connectivity Map Analysis Identifies Fisetin as a Treatment Compound for Osteoporosis Through Activating the PI3K-AKT Signaling Pathway in Mouse Pre-osteoblastic MC3T3-E1 Cells.

AIMS: This research aimed at exploring potential new compounds to be used in the treatment of osteoporosis by Connectivity Map (CMap) and determining the role of fisetin in osteoporosis according to its effects on the PI3K-AKT signaling pathway in MC3T3-E1 pre-osteoblastic cells. METHODS: Microarray analysis was used to obtain the differentially expressed genes in published gene expression data. Potent compounds for osteoporosis therapy were discovered by CMap analysis. DAVID and Gene Set Enrichment Analysis (GSEA) were used to discover signaling pathways that connected to osteoporosis disease. Cell viability was evaluated by a CCK-8 assay. Quantitative realtime Polymerase Chain Reaction (qRT-PCR) and western blot analysis were used to test the mRNA and protein expressions related to the PI3K-AKT signaling pathway in MC3T3-E1 cells, respectively. RESULTS: CMap analysis identified fisetin as a promising compound for anti-osteoporosis treatment. DAVID and GSEA analysis showed that the PI3K-AKT signaling pathway was inactivated in osteoporosis. Cell experiments revealed that fisetin caused an elevation of cell viability, up-regulated the mRNA levels of the Runt-related transcription factor-2 (Runx2), Osterix (Osx), collagen type I 1 (Col1a1) and Osteoprotegerin (OPG) while down-regulated the nuclear factor-κB ligand (RANKL) mRNA level. DISCUSSION: The protein levels of Runx2, Col1a1 and Osteocalcin (OCN) were also increased by fisetin. Furthermore, fisetin activated the phosphoinositide-3-kinase/protein kinase B (PI3K-AKT) signaling pathway, and blocking this pathway by the inhibitor LY-294002 could impair fisetin's functions on proliferation, differentiation and OPG/RANKL expression ratio in the MC3T3-E1 cells. CONCLUSION: Our results demonstrated that fisetin could promote MC3T3-E1 cell proliferation, differentiation and increase OPG/RANKL expression ratio through activating the PI3K-AKT pathway, which has potential for the treatment of osteoporosis.

Dihalocarbene insertion reactions into C-H bonds of compounds containing small rings: mechanisms and regio- and stereoselectivities.

Novel insertion reactions of dichloro- and dibromocarbene into carbon-hydrogen bonds adjacent to cyclopropane rings are reported. It is found that the predominant isomers formed in the reactions with bicyclo[4.1.0]heptane result from insertion into the endo carbon-hydrogen bonds alpha to the three-membered ring. In the reactions of bicyclo[3.1.0]hexane, however, the exo dihalocarbene insertion products are formed as the major isomers. In some compounds cyclopropane rings "activate" adjacent carbon-hydrogen bonds, whereas other systems containing three-membered rings do not. Moreover, the influence of various substituents (methyl, geminal dimethyl, phenyl, methoxy, and ethoxy) attached to bicyclo[3.1.0]hexane and bicyclo[4.1.0]heptane in dihalocarbene reactions has been studied. The findings can be explained by the concept of maximum orbital overlaps of Walsh orbitals of the cyclopropane rings and the alpha carbon-hydrogen bonds. In stark contrast, selective insertion into the tertiary carbon-hydrogen bonds of the cyclobutane ring in bicyclo[4.2.0]octane is observed.

Synthesis of releasable electrophore tags for applications in mass spectrometry.

Releasable electrophore mass tags (electrophore tags) are compounds for use as labels in ligand assays such as hybridization assays and immunoassays. In such assays, the electrophore-tagged reagent (e.g., DNA probe or antibody) is quantified at the conclusion of the assay by cleaving a bond in the attached tag so that the electrophore part can be brought into the gas phase (usually thermally) for detection by electron capture mass spectrometry (EC-MS) or a related technique. Interest in these tags is promoted mainly by their potential to provide highly sensitive and multiplexed assays. The high multiplexing arises from the opportunity to measure many such tags simultaneously in the mass spectrometer, where each tag has an electrophore part with a unique mass. In this study five precursors of electrophore mass tags are presented. Each precursor can lead to a large library of electrophore tags in a practical way, since each precursor can be converted to many different electrophore tags by reaction with commonly available phenols that provide a variation in mass. The phenol-reactive part of the tag is either a polyfluorobiphenyl or a benzyl chloride moiety. Representative library compounds are prepared and detected in an inert ester form by gas chromatography electron capture mass spectrometry (GC-EC-MS). Further, one tag is conjugated to DNA, and the resulting product is detected by laser-induced electron capture time-of-flight mass spectrometry on a silver surface. A calculation by the semiempirical method AM1 for an ion formed by one of the electrophores suggests that ring rotation promotes dissociative electron capture. The features of practical synthesis, simple composition, physicochemical stability, high multiplicity, high sensitivity, and potential for high throughput detection make releasable electrophore mass tags attractive for highly multiplexed assays. This includes their use in SNP assays or dideoxy DNA sequencing for detection of mutations in individuals, where the combination of high accuracy and speed is essential.

Genetic interaction between calcineurin and type 2 myosin and their involvement in the regulation of cytokinesis and chloride ion homeostasis in fission yeast.

Calcineurin plays a critical role in Ca(2+) signaling in various cell types. In fission yeast, calcineurin is required for cytokinesis and chloride ion homeostasis. However, most of its physiological functions remain obscure. A genetic screen was performed to identify genes that share an essential function with calcineurin. We screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and to a high concentration of chloride ion and isolated a mutant, cis2-1/myp2-c2, which contains a novel allele of the myp2(+)/myo3(+) gene that encodes a type 2 myosin heavy chain. The myp2-c2 mutant showed morphological defects similar to those associated with a calcineurin deletion mutant, such as multiseptated and branched cells. Consistently, myp2-null cells were hypersensitive to chloride ion and showed the multiseptated phenotype in the presence of immunosuppressants or at high chloride concentrations. Overexpression of constitutively active calcineurin suppressed the chloride ion-sensitive growth defect and cytokinesis abnormality of the myp2-c2 mutant and myp2-null cells. Interestingly, the essential myosin light chain mutant cdc4-8 failed to grow and could not form a normal contractile ring in the presence of immunosuppressants. Furthermore, calcineurin-null cells exhibited aberrant contractile rings, suggesting impaired contraction of the rings. These results indicate that calcineurin is involved in the regulation of cytokinesis and that chloride ion homeostasis is mediated by type 2 myosin.