Three-dimensional (3D)-printing techniques such as stereolithography (SLA) are currently gaining momentum for the production of miniaturized analytical devices and molds for soft lithography. However, most commercially available SLA resins inhibit polydimethylsiloxane (PDMS) curing, impeding reliable replication of the 3D-printed structures in this elastomeric material. Here, we report a systematic study, using 16 commercial resins, to identify a fast and straightforward treatment of 3D-printed structures and to support accurate PDMS replication using UV and/or thermal post-curing. In-depth analysis using Raman spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry revealed that phosphine oxide-based photo-initiators, leaching out of the 3D-printed structures, are poisoning the Pt-based PDMS catalyst. Yet, upon UV and/or thermal treatments, photo-initiators were both eliminated and recombined into high molecular weight species that were sequestered in the molds.

Starch bound proteins mainly include enzymes from the starch biosynthesis pathway. Recently, new functions in starch molecular assembly or active protein targeting were also proposed for starch associated proteins. The potato genome sequence reveals 77 loci encoding starch metabolizing enzymes with the identification of previously unknown putative isoforms. Here we show by bottom-up proteomics that most of the starch biosynthetic enzymes in potato remain associated with starch even after washing with SDS or protease treatment of the granule surface. Moreover, our study confirmed the presence of PTST1 (Protein Targeting to Starch), ESV1 (Early StarVation1) and LESV (Like ESV), that have recently been identified in Arabidopsis. In addition, we report on the presence of a new isoform of starch synthase, SS6, containing both K-X-G-G-L catalytic motifs. Furthermore, multiple protease inhibitors were also identified that are cleared away from starch by SDS and thermolysin treatments. Our results indicate that SS6 may play a yet uncharacterized function in starch biosynthesis and open new perspectives both in understanding storage starch metabolism as well as breeding improved potato lines.

The methacrylamide-modified recombinant peptide (RCPhC1-MA) is an attractive synthetic alternative for animal-derived Gel-MA.

Polymer engineering, such as in three-dimensional (3D) printing, is rapidly gaining popularity, not only in the scientific and medical fields but also in the community in general. However, little is known about the toxicity of engineered materials. Therefore, we assessed the toxicity of 3D-printed and molded parts from five different polymers commonly used for prototyping, fabrication of organ-on-a-chip platforms, and medical devices. Toxic effects of PIC100, E-Shell200, E-Shell300, polydimethylsiloxane, and polystyrene (PS) on early bovine embryo development, on the transactivation of estrogen receptors were assessed, and possible polymer-leached components were identified by mass spectrometry. Embryo development beyond the two-cell stage was inhibited by PIC100, E-Shell200, and E-Shell300 and correlated to the released amount of diethyl phthalate and polyethylene glycol. Furthermore, all polymers (except PS) induced estrogen receptor transactivation. The released materials from PIC100 inhibited embryo cleavage across a confluent monolayer culture of oviduct epithelial cells and also inhibited oocyte maturation. These findings highlight the need for cautious use of engineered polymers for household 3D printing and bioengineering of culture and medical devices and the need for the safe disposal of used devices and associated waste.

Obtaining the full MS/MS map for fragments and precursors of complex mixtures without hyphenation with chromatographic separation by a data-independent acquisition is a challenge in mass spectrometry which is solved by two-dimensional (2D) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Until now 2D FTICR MS afforded only a moderate resolution for precursor ion since this resolution is limited by the number of evolution interval steps to which the number of scans, the acquisition time, and the sample consumption are proportional. An overnight acquisition is already required to reach a quadrupole mass filter-like unit mass resolution. Here, we report that 2D FTICR MS using nonuniform sampling (NUS) obtained by randomly skipping points in the first dimension corresponding to the precursor selection gives access, after data processing, to the same structural information contained in a complex mixture. The resolution increases roughly as the inverse of the NUS ratio, up to 26 times at NUS 1/32, leading to an acquisition time reduced in the same ratio compared to a classical acquisition at the same resolution. As an example, the analysis of a natural oil is presented.

Various biopolymers, including gelatin, have already been applied to serve a plethora of tissue engineering purposes. However, substantial concerns have arisen related to the safety and the reproducibility of these materials due to their animal origin and the risk associated with pathogen transmission as well as batch-to-batch variations. Therefore, researchers have been focusing their attention toward recombinant materials that can be produced in a laboratory with full reproducibility and can be designed according to specific needs (e.g., by introducing additional RGD sequences). In the present study, a recombinant protein based on collagen type I (RCPhC1) was functionalized with photo-cross-linkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), or thiol (RCPhC1-SH) functionalities to enable high-resolution 3D printing via two-photon polymerization (2PP). The results indicated a clear difference in 2PP processing capabilities between the chain-growth-polymerized RCPhC1-MA and the step-growth-polymerized RCPhC1-NB/SH. More specifically, reduced swelling-related deformations resulting in a superior CAD-CAM mimicry were obtained for the RCPhC1-NB/SH hydrogels. In addition, RCPhC1-NB/SH allowed the processing of the material in the presence of adipose tissue–derived stem cells that survived the encapsulation process and also were able to proliferate when embedded in the printed structures. As a consequence, it is the first time that successful HD bioprinting with cell encapsulation is reported for recombinant hydrogel bioinks. Therefore, these results can be a stepping stone toward various tissue engineering applications.

SignificanceUsing an interactomic approach, we have identified the nuclear receptor REV-ERBα as a O-GlcNAc transferase (OGT) protein partner. REV-ERBα protects cytoplasmic OGT from proteasomal degradation and facilitates cytosolic and nuclear protein O-GlcNAcylation while REV-ERα ligands decreased cytoplasmic OGT activity. REV-ERBα thus exerts pleiotropic activities through OGT, coordinating signal transduction, epigenomic programming, and transcriptional response in the liver.

O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT’s role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.