ListOfExperiments+FitParam

This Excel file lists all the experiments with the conditions for each recording, and the resulting parameters that were obtained after fitting the NLC and the electromotility curve (when applicable)

OHC_Dimension only

This file contains the pictures of OHCs that were not subjected to diflunisal treatment, and used only to obtain dimension data

OHC_NLC&eM

These are the data for the outer hair cell. Files are grouped by date and experiment number (refer to the list of experiments) The electrophysiology data are provided in HEKA format as well as text format. The videos of the electromotility are provided in as Tif files. The analysis of the electromotility is also included as individual Excel files for each recording

HEK_NLC recordings

Electrophysiology data for HEKs expressing prestin, in the absence and presence of diflunisal (refer to the list of experiments). The original file is .dat for HEKA analysis software. Files are also provided in a text format

NLC and eM in the presence of DFL and in different chloride conditions.

<p><b>(A)</b> V_1/2 value obtained with OHCs from two-state Boltzmann fits of the NLC (○ at low & ● at high intracellular chloride) and the eM (◻ at low & at high intracellular chloride), plotted against DFL concentration. <b>(B)</b> The difference V1/2(NLC)—V_1/2(eM) shows decoupling between charge transfer and electromotility as [DFL] increases. The t-test is conducted between the values obtained without DFL versus with DFL * p<0.01; ** p<0.001. <b>(C)</b> Inhibition of the NLC and <b>(D)</b> of the eM by DFL. The dotted line is the resulting fit of the data by a Hill equation (140mM Cl: n_H = -0.66, IC₅₀ = 331 μM; 5mM Cl^-: n_H = -1.12, IC₅₀ = 98 μM), The resulting eM is only inhibited at concentrations above 0.5 mM in low chloride conditions. The value for eM at 1mM DFL at low chloride concentration was obtained from direct measurement of cell length at hyperpolarized and depolarized voltages (star). For all plots, the data points represent the average and the error bar is the standard deviation for n≥3 cells per condition.</p

Diflunisal inhibits prestin by chloride-dependent mechanism

<div><p>The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride <i>independent</i> increase in the surface area and the specific capacitance of the membrane, and a chloride <i>dependent</i> inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.</p></div

DFL affects the capacitance and the length of the OHC.

<p><b>(A)</b> The effect of DFL on absolute C_lin and C_max values and <b>(B)</b> relative C_lin (compiled on a cell to cell basis) are compared to <b>(C)</b> the change in total cell length, plotted as the ratio of OHC length with DFL to without. In order to have reliable data at high DFL concentrations, we used the fully extended length to calculate this ratio. <b>(D)</b> Specific capacitance C_lin/A (pF/μm²) is calculated for each OHC and showed in 3 distribution histograms. The diameter and length of each cell was measured and the resulting area was determined. Low DFL: [DFL]≤10⁻² mM & High DFL: [DFL]>10⁻² mM. The averages obtained from the probability distribution fitting of the raw data (grey line) are 9.45±1.9, 9.68±1.7 and 10.97±2.1 fF/μm². (<b>E</b>) The surface area specific capacitance C_SA as well as <b>(F)</b> the difference between C_SA and C_lin (ΔC_SA) are obtained from fitting the NLCs. ΔC_SA is normalized by dividing by the number of active motors <i>N</i> in the absence of drug on a cell to cell basis. Each value is averaged from 3 independent recordings or more, and the error bars represent the standard deviation. (t-test: * p<0.01; ** p<0.001).</p

Synthesis of Cyclopropanated [2.2.1] Heterobicycloalkenes: An Improved Procedure

<div><p></p><p>A safer and improved method to our previous report on palladium-catalyzed cyclopropanation of heterobicyclic alkenes has been developed. By using THF as the solvent and a more dilute aqueous NaOH solution for the generation of diazomethane from Diazald®, cyclopropanation could be achieved smoothly with minimal adjustment over the course of reaction. 7-Oxabicyclic substrates with bulky C1 or C2 groups, as well as 2,3-diazabicyclic substrates with various N-substituents effectively underwent cyclopropanation. Using this methodology, yields to previously reported products were markedly increased, and 10 new cyclopropanated [2.2.1] heterobicyclic products were prepared. In addition, this work accounts for the first reported cyclopropanation of 2,3-diazabicyclic alkenes, which all gave excellent yields of >90%.</p></div

Intramolecular Inverse Electron-Demand [4 + 2] Cycloadditions of Ynamides with Pyrimidines: Scope and Density Functional Theory Insights

4-Aminopyridines are valuable scaffolds for the chemical industry in general, from life sciences to catalysis. We report herein a collection of structurally diverse polycyclic fused and spiro-4-aminopyridines that are prepared in only three steps from commercially available pyrimidines. The key step of this short sequence is a [4 + 2]/<i>retro</i>-[4 + 2] cycloaddition between a pyrimidine and an ynamide, which constitutes the first examples of ynamides behaving as electron-rich dienophiles in [4 + 2] cycloaddition reactions. In addition, running the <i>ih</i>DA/<i>r</i>DA reaction in continuous mode in superheated toluene, to overcome the limited scalability of MW reactions, results in a notable production increase compared to batch mode. Finally, density functional theory investigations shed light on the energetic and geometric requirements of the different steps of the <i>ih</i>DA/<i>r</i>DA sequence