Coumarin-Based Oxime Esters: Photobleachable and Versatile Unimolecular Initiators for Acrylate and Thiol-Based Click Photopolymerization under Visible Light-Emitting Diode Light Irradiation

Developing efficient unimolecular visible light-emitting diode (LED) light photoinitiators (PIs) with photobleaching capability, which are essential for various biomedical applications and photopolymerization of thick materials, remains a great challenge. Herein, we demonstrate the synthesis of a series of novel PIs, containing coumarin moieties as chromophores and oxime ester groups as initiation functionalities and explore their structure–activity relationship. The investigated oxime esters can effectively induce acrylates and thiol-based click photopolymerization under 450 nm visible LED light irradiation. The initiator <b>O-3</b> exhibited excellent photobleaching capability and enabled photopolymerization of thick materials (∼4.8 mm). The efficient unimolecular photobleachable initiators show great potential in dental materials and 3D printings

Fine Mapping of a Degenerated Abdominal Legs Mutant (<i>E^dl</i>) in Silkworm, <i>Bombyx mori</i>

<div><p>In insects, abdominal appendages, also called prolegs, vary due to adaptive evolution. Mutations on prolegs within species provide insights to better understand the mechanisms underlying appendage development and diversity. In silkworm <i>Bombyx mori</i>, extra-crescents and degenerated abdominal legs (<i>E</i>^<i>dl</i>) mutant, belonging to the <i>E</i> pseudoallele group, is a spontaneous mutation that adds crescents and degenerates prolegs on the third abdominal segment (A3). This mutation may be a homeotic transformation of A3 to A2. In this study, the <i>E</i>^<i>dl</i> locus was mapped within approximately a 211 Kb region that is 10 Kb upstream of <i>Bmabdominal-A</i> (<i>Bmabd-A</i>). RT-quantitative PCR (RT-qPCR) and Western blot analysis of <i>Bmabd-A</i> expression showed a slight but significant decrease, while the expression of <i>BmUltrabithorax</i> (<i>BmUbx</i>) was up-regulated in the <i>E</i>^<i>dl</i> mutant compared to wildtype (Dazao). Moreover, we also found that BmDistal-less (BmDll), which regulated the development of distal proleg structures, was missing at the tips of the A3 prolegs in the <i>E</i>^<i>dl</i> mutant compared to BmDll expression in normally developed prolegs in both the wildtype and mutant. Collectively, we identified approximately a 211 Kb region in the <i>E</i>^<i>dl</i> locus that regulates <i>BmUbx</i> and <i>Bmabd-A</i> expression and found that changes in <i>BmUbx</i> and <i>Bmabd-A</i> expression may lead to the loss of distal proleg structures in <i>B</i>. <i>mori</i>.</p></div

Sequences of primers used in this study.

<p>Sequences of primers used in this study.</p

Phenotype of wildtype (Dazao) and extra-crescents and degenerated abdominal legs (<i>E</i>^<i>dl</i>) mutant.

<p>Lateral views of embryos (A), and dorsal and lateral views of larvae (B, C) are shown with head on the left side. (A, B) Prolegs on the third abdominal segment (A3) are degenerated in <i>E</i>^<i>dl</i> compared to those in Dazao. Blue arrowheads indicate prolegs on A3 in <i>E</i>^<i>dl</i> and Dazao. (C) Extra crescents are arising on the dorsal side of A3 in <i>E</i>^<i>dl</i>. Blue arrowheads indicate the marking in the dorsal part of A3 in <i>E</i>^<i>dl</i> and Dazao. Yellow arrowheads indicate the marking in the dorsal part of A8 in <i>E</i>^<i>dl</i>. Bars A 1 mm; B, C 1 cm.</p

<i>Bombyx</i> Distal-less (BmDll) antibody staining of embryos at stage 20 in Dazao and <i>E</i>^<i>dl</i> mutant.

<p>BmDll staining (red) was detected in the distal part of all developed appendage primordia. T1, T2 and T3 represent three thoracic segments. The image was focused on abdominal appendages. Blue arrowheads were used for marking the prolegs on A3 segment. (A) BmDll staining was detected in the distal part of prolegs on A3-A6 and A10 segments in Dazao. (B) BmDll staining was not detected in the distal part of prolegs on A3 and was normally detected on A4-A6 and A10 segments in <i>E</i>^<i>dl</i>. The prolegs on A3 were also degenerated compared to prolegs on A4-A6 in <i>E</i>^<i>dl</i>.</p

Expression of <i>BmUbx</i> and <i>Bmabd-A</i> in wildtype and <i>E</i>^<i>dl</i> mutant in stage 20 embryos.

<p>(A, C) RT-qPCR analysis of <i>Bmabd-A</i> and <i>BmUbx</i>. <i>Bmabd-A</i> was significantly down-regulated and <i>BmUbx</i> was up-regulated in <i>E</i>^<i>dl</i> mutants (Bars indicate mean values ± SD, Student’s <i>t</i>-test. *, P<0.05. n = 3). SW2934 was used as the internal control. (B) Western blot analysis of Bmabd-A. Expression level of Bmabd-A protein was decreased in <i>E</i>^<i>dl</i> mutants. Tubulin was used as the control to monitor equal loading of total proteins.</p

Laser Photofabrication of Cell-Containing Hydrogel Constructs

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays

A Straightforward Synthesis and Structure–Activity Relationship of Highly Efficient Initiators for Two-Photon Polymerization

The development of practical two-photon absorption photoinitiators (TPA PIs) has been slow due to their complicated syntheses often reliant on expensive catalysts. These shortcomings have been a critical obstruction for further advances in the promising field of two-photon-induced photopolymerization (TPIP) technology. This paper describes a series of linear and cyclic benzylidene ketone-based two-photon initiators containing double bonds and dialkylamino groups synthesized in one step via classical aldol condensation reactions. Systematic investigations of structure–activity relationships were conducted via quantum-chemical calculations and experimental tests. These results showed that the size of the central ring significantly affected the excited state energetics and emission quantum yields as well as the two-photon initiation efficiency. In the TPIP tests the 4-methylcyclohexanone-based initiator displayed much broader ideal processing windows than its counterparts with a central five-membered ring and previously described highly active TPA PIs. Surprisingly, a writing speed as high as 80 mm/s was obtained for the microfabrication of complex 3D structures employing acrylate-based formulations. These highly active TPA PIs also exhibit excellent thermal stability and remain inert to one-photon excitation. Straightforward synthesis combined with high TPA initiation efficiency makes these novel initiators promising candidates for commercialization

A Straightforward Synthesis and Structure–Activity Relationship of Highly Efficient Initiators for Two-Photon Polymerization

The development of practical two-photon absorption photoinitiators (TPA PIs) has been slow due to their complicated syntheses often reliant on expensive catalysts. These shortcomings have been a critical obstruction for further advances in the promising field of two-photon-induced photopolymerization (TPIP) technology. This paper describes a series of linear and cyclic benzylidene ketone-based two-photon initiators containing double bonds and dialkylamino groups synthesized in one step via classical aldol condensation reactions. Systematic investigations of structure–activity relationships were conducted via quantum-chemical calculations and experimental tests. These results showed that the size of the central ring significantly affected the excited state energetics and emission quantum yields as well as the two-photon initiation efficiency. In the TPIP tests the 4-methylcyclohexanone-based initiator displayed much broader ideal processing windows than its counterparts with a central five-membered ring and previously described highly active TPA PIs. Surprisingly, a writing speed as high as 80 mm/s was obtained for the microfabrication of complex 3D structures employing acrylate-based formulations. These highly active TPA PIs also exhibit excellent thermal stability and remain inert to one-photon excitation. Straightforward synthesis combined with high TPA initiation efficiency makes these novel initiators promising candidates for commercialization