No abstract

We have generated the first mouse model of fibro-blast growth factor receptor 3 (Fgfr3) with the K644E mutation, which accurately reflects the embryonic onset of a neonatal lethal dwarfism, thanatophoric dysplasia type II (TDII). Long-bone abnormalities were identified as early as embryonic day 14, during initiation of endochondral ossification. Increased expression of PATCHED: (PTC:) was observed, independent of unaltered expression of parathyroid hormone-related peptide (PTHrP) receptor and Indian Hedgehog (IHH:), suggesting a new regulatory role for Fgfr3 in embryos. We demonstrate that the mutation enhances chondrocyte proliferation during the early embryonic skeletal development, in contrast to previous reports that showed decreased proliferation in postnatal-onset dwarf mice with activating Fgfr3 mutations. This suggests that signaling through Fgfr3 both promotes and inhibits chondrocyte proliferation, depending on the time during development. In contrast, suppressed chondrocyte differentiation was observed throughout the embryonic stages, defining decreased differentiation as the primary cause of retarded longitudinal bone growth in TDII. This model was successfully crossed with a cartilage-specific CRE: transgenic strain, excluding the lung as the primary cause of lethality.

Coronary intervention following ST-segment elevation myocardial infarction (STEMI) is the treatment of choice for reducing cardiomyocyte death but paradoxically leads to reperfusion injury. Pharmacological post-conditioning is an attractive approach to minimize Ischemia-Reperfusion Injury (IRI), but candidate drugs identified in IRI animal models have performed poorly in human clinical trials, highlighting the need for a human cell-based model of IRI. In this work, we show that when we imposed sequential hypoxia and reoxygenation episodes [mimicking the ischemia (I) and reperfusion (R) events] to immature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), they display significant hypoxia resistance and minimal cell death (∼5%). Metabolic maturation of hPSC-CMs for 8 days substantially increased their sensitivity to changes in oxygen concentration and led to up to ∼30% cell death post-hypoxia and reoxygenation. To mimic the known transient changes in the interstitial tissue microenvironment during an IRI event in vivo , we tested a new in vitro IRI model protocol that required glucose availability and lowering of media pH during the ischemic episode, resulting in a significant increase in cell death in vitro (∼60%). Finally, we confirm that in this new physiologically matched IRI in vitro model, pharmacological post-conditioning reduces reperfusion-induced hPSC-CM cell death by 50%. Our results indicate that in recapitulating key aspects of an in vivo IRI event, our in vitro model can serve as a useful method for the study of IRI and the validation and screening of human specific pharmacological post-conditioning drug candidates.

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch’s t-test returning P < 0.05 and Mann–Whitney test P < 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P < 0.01) and 0.63 (P < 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC.

Radical anions (RAs) are the key intermediates of the selective hydrodefluorination of polyfluoroarenes. We used the techniques of optically detected electron paramagnetic resonance (OD EPR), time-resolved fluorescence, time-resolved magnetic field effect (TR MFE), and the density functional theory to study the possibility of RAs formation from 4-aminononafluorobiphenyl (1) and pentafluoroaniline (2) and estimate their lifetimes and decay channels. To our knowledge, both RAs have not been detected earlier. We have registered the OD EPR spectrum for relatively stable in nonpolar solutions 1(-•) but failed to register the spectra for 2(-•). However, we have managed to fix the 2(-•) by the TR MFE method and obtained its hyperfine coupling constants. The lifetime of 2(-•) was found to be only a few nanoseconds. The activation energy of its decay was estimated to be 3.6 ± 0.3 kcal/mol. According to the calculation results, the short lifetime of 2(-•) is due to the RA fast fragmentation with the F(-) elimination from ortho-position to the amine group. The calculated energy barrier, 3.2 kcal/mol, is close to the experimental value. The fragmentation of 2(-•) in a nonpolar solvent is possible due to the stabilization of the incipient F(-) anion by the binding with the amine group proton.