Supplementary document for Broadband achromatic and wide field-of-view single-layer metalenses in the mid-infrared - 6648865.pdf

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Supplementary document for Broadband achromatic and wide field-of-view single-layer metalenses in the mid-infrared - 6641886.pdf

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Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold – A Potential Therapeutic Intervention for Skin Diseases

<div><p>Tissue kallikreins (KLKs), in particular KLK5, 7 and 14 are the major serine proteases in the skin responsible for skin shedding and activation of inflammatory cell signaling. In the normal skin, their activities are controlled by an endogenous protein protease inhibitor encoded by the <i>SPINK5</i> gene. Loss-of-function mutations in <i>SPINK5</i> leads to enhanced skin kallikrein activities and cause the skin disease Netherton Syndrome (NS). We have been developing inhibitors based on the Sunflower Trypsin Inhibitor 1 (SFTI-1) scaffold, a 14 amino acids head-to-tail bicyclic peptide with a disulfide bond. To optimize a previously reported SFTI-1 analogue (I10H), we made five analogues with additional substitutions, two of which showed improved inhibition. We then combined those substitutions and discovered a variant (Analogue 6) that displayed dual inhibition of KLK5 (tryptic) and KLK7 (chymotryptic). Analogue 6 attained a tenfold increase in KLK5 inhibition potency with an Isothermal Titration Calorimetry (ITC) K_d of 20nM. Furthermore, it selectively inhibits KLK5 and KLK14 over seven other serine proteases. Its biological function was ascertained by full suppression of KLK5-induced Protease-Activated Receptor 2 (PAR-2) dependent intracellular calcium mobilization and postponement of Interleukin-8 (IL-8) secretion in cell model. Moreover, Analogue 6 permeates through the cornified layer of <i>in vitro</i> organotypic skin equivalent culture and inhibits protease activities therein, providing a potential drug lead for the treatment of NS.</p></div

Control of IL-8 Secretion by Analogue 6 in KLK5 Simulated N-tert Skin Cells.

<p>Changes in IL-8 level in the cell medium was monitored 6, 24 and 48 hours after identical cell populations were stimulated with fresh medium (Medium Only), or KLK5 (KLK5 Only, f/c 300 nM), or KLK5 + Analogue 6 (KLK5 + Analogue 6), or Analogue 6 (Analogue 6 Only, f/c 1515 nM), or 0.2 mM MES (Medium + 0.2 mM MES). All substances were diluted with cell culture medium. Control samples including the medium with MES were used to ensure that MES which was in the KLK5 protein stock solution did not cause IL-8 level to change. Basal IL-8 level prior to stimulation (0hr pre-treatment) as well as for unstimulated cells 48hrs after (48hr untreated) were also determined to serve as quality controls. A coefficient variation of around 11% and 6% for 0hr pre-treatment and 48hr untreated respectively indicates the status of cell populations in each well was homogenous throughout the experiment. Error bars are standard deviations of IL-8 readings from different wells of cells. N.S. Not Statistically Significant; * p<0.05; **p<0.01.</p

Suppression of KLK5-Induced Intracellular Calcium Mobilization by Native SFTI and Its Analogues.

<p>Change in relative fluorescence unit (ΔRFU) was monitored continuously for about 5 minutes after injection of a mixture of KLK5 (774 nM) with various concentrations of native SFTI or its analogues. KLK5 and the inhibitors were diluted in 1xPBS and the concentration values indicate their final concentration in the well. For each tested ligand, a positive control (KLK5 Only) and negative control (Inhibitor Only) were performed to ensure the inhibitor and the diluent does not cause a signal. Total suppression of KLK5-induced intracellular calcium mobilization was achieved at an inhibitor-to-enzyme ratio of 2:1 for native SFTI (A), 4:1 for I10H (B.) and 1:1 for Analogue 2 (C) and Analogue 6 (D).</p

Regions Targeted by I10H Analogues Outside of the KLK5 Substrate Binding Pocket.

<p>Molecular docking has revealed several regions (blue, green and red) of KLK5 outside the substrate specificity binding pocket (magenta) that SFTI-1 (shown in cylinders) could engage for extended specific interactions. Surface display of the model was generated by CCP4MG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166268#pone.0166268.ref034" target="_blank">34</a>].</p

Chemical Structure of Sunflower Trypsin Inhibitor-1 and the “HC” Synthetic Strategy.

<p>Chemical structure of native SFTI (<b>A</b>) and the synthetic strategy for “HC” analogue synthesis (<b>B</b>) are shown. Amino acid residues and their position in the native peptide sequence are labelled in letters and numbers respectively. The P1 residue which will fit into the enzyme substrate binding pocket is lysine located at position 5 with the scissile bond indicated by an arrow. The side of SFTI-1 that forms direct contact with the enzyme and the non-contacting side are segregated by the red line.</p

Ternary Photoanodes with AgAu Nanoclusters and CoNi-LDH for Enhanced Photoelectrochemical Water Oxidation

Atomically precise metal nanoclusters (NCs) present new opportunities for creating innovative solar-powered photoanodes due to their extraordinary physicochemical properties. Nevertheless, ultrasmall metal NCs tend to aggregate and lack active sites under light irradiation, which severely limits their widespread application. We have developed a strategy to design efficient ternary photoanodes by successively modifying AgAu NCs and CoNi-LDH on BiVO4 substrates using versatile impregnation and electrodeposition. The electronic properties of AgAu NCs facilitate the rapid transfer of photogenerated carriers on BiVO4 and CoNi-LDH. Additionally, ultrathin CoNi-LDH acts as a hole-collecting layer, which quickly extracts holes to the electrode/electrolyte interface. The synergistic effect and the matched energy levels between the ternary heterostructures promote the OER process, which significantly improved the photoelectrochemical (PEC) water oxidation performance. This study presents a new idea for further exploration of metal nanocluster-based PEC systems

Enzyme Inhibitory Profile of I10H Analogues.

<p>Enzyme Inhibitory Profile of I10H Analogues.</p

Fitted IC₅₀ Curves of KLK5 Inhibition by Native SFTI and Its Analogues.

<p>The fitted KLK5 inhibition IC₅₀ curves including error bars (standard deviation) of each repeated reading (N = 3) for SFTI-1 and its analogue are shown.</p