Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is
ARL2BP
, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in
ARL2BP
in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy.
Stable tetrathiatriarylmethyl
radicals have significantly contributed to the recent progress in
biomedical electron paramagnetic resonance (EPR) due to their unmatched
stability in biological media and long relaxation times. However,
the lipophilic core of the most commonly used structure (Finland trityl)
is responsible for its interaction with plasma biomacromolecules,
such as albumin, and self-aggregation at high concentrations and/or
low pH. While Finland trityl is generally considered inert toward
many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl
with a high rate constant of 3.53 × 108 M–1 s–1, leading to a trisulfonated Finland trityl
radical. This newly synthesized highly hydrophilic trityl radical
shows an ultranarrow linewidth (ΔB
pp = 24 mG), a lower affinity for albumin than Finland trityl, and
a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl
radical can be considered as a superior spin probe in comparison to
the widely used Finland trityl. One of its potential applications
was demonstrated by in vivo mapping oxygen in a mouse
model of breast cancer. Moreover, we showed that one of the three
sulfo groups can be easily substituted with S-, N-, and P-nucleophiles,
opening access to various monofunctionalized sulfonated trityl radicals.
Enzyme activities are well established biomarkers of many pathologies. Imaging enzyme activity directly in vivo may help to gain insight into the pathogenesis of various diseases but remains extremely challenging. In this communication, we report the use of EPR imaging (EPRI) in combination with a specially designed paramagnetic enzymatic substrate to map alkaline phosphatase activity with a high selectivity, thereby demonstrating the potential of EPRI to map enzyme activity.
The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO 2 at the active site of PO followed by oxidation of DOPA by NO 2 . On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect's haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.
Enzyme activities are well established biomarkers of many pathologies. Imaging enzyme activity directly in vivo may help to gain insight into the pathogenesis of various diseases but remains extremely challenging. In this communication, we report the use of EPR imaging (EPRI) in combination with a specially designed paramagnetic enzymatic substrate to map alkaline phosphatase activity with a high selectivity, thereby demonstrating the potential of EPRI to map enzyme activity.
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