Selective Targeting of Class I Histone Deacetylases in a Model of Human Osteosarcoma.

Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.

Precise detection of a murine germline mutation of the Notch3 gene associated with kyphosis and developmental disorders.

OBJECTIVE: Humpback (hpbk) mice harbor a pathogenic mutation in the Notch3 gene and can serve as a beneficial animal model for investigating human myopathy, kyphosis, and developmental disorders, including lateral meningocele syndrome. Detection of the point mutation in hpbk mice is important for maintaining strains and scrutinizing genetic rescues, especially considering that homozygous mice are infertile and indistinguishable from their littermates at a young age. This study aimed for the development of a novel, precise, and time-saving genotyping method to identify the mutation in hpbk mice. MATERIALS AND METHODS: In order to study the hpbk mouse line, we describe how we applied several tools, including quantitative polymerase chain reaction (qPCR), multiplex tetra-primer amplification-refractory mutation system (ARMS-PCR) and Sanger sequencing, toward the recognition of heterozygous and homozygous mice. RESULTS: The Notch3 mutation was clearly identified using qPCR and ARMS assays, but the latter was a more precise and cost-effective approach. The lengths of the ARMS-PCR amplicons are 210 bp and 164 bp for the wild-type and hpbk alleles, respectively. Moreover, the genotyping results for each mouse were corroborated by Sanger DNA sequencing. CONCLUSION: Our newly developed PCR-based ARMS system affords a swift and precise way to genotype the hpbk mice. ARMS-PCR does not rely on any advanced equipment and is useful as a genotyping method for other model organisms that harbor a pathogenic variant.

Chigger mites of the genus Ericotrombidium (Acariformes: Trombiculidae) attacking pets in Europe.

Diversity of chigger mites causing trombiculiasis of domestic animals and humans in Europe is greatly underestimated. A number of reports on the attacks of "harvest mite" (Neotrombicula autumnalis) could be based on misidentified chiggers from other species and genera. In this study descriptions of two cases of trombiculiasis are presented, which constitute the first report on the pets' parasitism by the chigger genus Ericotrombidium in Europe. The species Ericotrombidium ibericense is for the first time reported in Portugal as a causative agent of the trombiculiasis entailed extensive alopecic lesions and pruritus in a cat. Ericotrombidium geloti is for the first time reported as a cause of canine trombiculiasis in Crimea. Presence of other Ericotrombidium species on man and domestic animals is highly probable in countries of the Mediterranean basin.

In vitro inhibition of the activation of Pro-matrix Metalloproteinase 1 (Pro-MMP-1) and Pro-matrix metalloproteinase 9 (Pro-MMP-9) by rice and soybean Bowman-Birk inhibitors.

The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI) or soybean Bowman-Birk inhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 microg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 microg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated that pro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient.