Protein Hydrogels with Reversibly Patterned Multidimensional Fluorescent Images for Information Storage.

Fluorescent polymeric hydrogels are promising soft and wet media for information storage that are desirable for lifelike biomaterials and flexible electronics. Hydrogels based on engineered proteins have attracted considerable interest. However, their potential utility as information storage media has remained largely unexplored. Here, we report a protein-based hydrogel that can serve as an information storage medium. Using LOVTRAP, which consists of protein LOV2 and its binding partner ZDark1, we developed a novel strategy to decorate/release fluorescent proteins onto/from a blank protein hydrogel slate in light-controlled and spatially defined fashions, reversibly generating fluorescent patterns such as quick response codes. To increase the information storage capacity, we further developed grayscale patterning to generate pseudo-colored multi-dimensional fluorescent images. Results of this new method demonstrate a novel reversible information storage approach in soft and wet materials and open a new avenue toward developing next-generation protein-based smart materials for information storage and anti-counterfeit applications.

Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP.

Protein-based hydrogels can mimic many aspects of native extracellular matrices (ECMs) and are promising biomedical materials that find various applications in cell proliferation, drug/cell delivery, and tissue engineering. To be adapted for different tasks, it is important that the mechanical and/or biochemical properties of protein-based hydrogels can be regulated by external stimuli. Light as a regulation stimulus is of advantage because it can be easily applied in demanded spatiotemporal manners. The noncovalent binding between the light-oxygen-voltage-sensing domain 2 (LOV2) and its binding partner ZDark1 (zdk1), named as LOVTRAP, is a light-responsive interaction. The binding affinity of LOVTRAP is much higher in dark than that under blue light irradiation. Taking advantage of these light-responsive interactions, herein we endeavored to use LOVTRAP as a crosslinking mechanism to engineer light-responsive protein hydrogels. Using LOV2-containing and zdk1-containing multifunctional protein building blocks, we successfully engineered a light-responsive protein hydrogel whose viscoelastic properties can change in response to light: in the dark, the hydrogel showed higher storage modulus; under blue light irradiation, the storage modulus decreased. Due to the noncovalent nature of the LOVTRAP, the engineered LOVTRAP protein hydrogels displayed shear-thinning and self-healing properties and served as an excellent injectable protein hydrogel. We anticipated that this new class of light-responsive protein hydrogels will broaden the scope of dynamic protein hydrogels and help develop other light-responsive protein hydrogels for biomedical applications.

Overexpression of abnormal spindle-like microcephaly-associated (ASPM) increases tumor aggressiveness and predicts poor outcome in patients with lung adenocarcinoma.

BACKGROUND: Cumulative evidence points to abnormal spindle-like microcephaly-associated (ASPM) protein being overexpressed in various cancers, and the aberrant expression of ASPM has been shown to promote cancer tumorigenicity and progression. However, its role and clinical significance in lung adenocarcinoma (LUAD) remains unclear. This study aimed to determine the expression patterns of ASPM and its clinical significance in LUAD. METHODS: In total, 4 original worldwide LUAD microarray mRNA expression datasets (N=1,116) with clinical and follow-up annotations were downloaded from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. The expression of ASPM protein in LUAD patients was detected by immunohistochemistry. Survival analysis and Cox regression analysis were used to examine the prognostic value of ASPM expression. Gene set enrichment analysis (GSEA) was performed to investigate the relationship between ASPM and LUAD. RESULTS: Dataset analyses and immunohistochemistry revealed that ASPM expression was significantly higher in the LUAD tissues compared with normal lung tissues, especially in the advanced tumor stage. Additionally, overexpression of ASPM was significantly correlated with shorter overall survival (OS) and relapse-free survival (RFS) in LUAD. Univariate and multivariate Cox regression analyses revealed that the overexpression of ASPM was a potential independent predictor of poor OS and RFS. However, ASPM overexpression was not significantly associated with predicting OS in lung squamous cell carcinoma. GSEA analysis demonstrated that ASPM was significantly enriched in the cell cycle, DNA replication, homologous recombination, RNA degradation, mismatch repair, and p53 signaling pathways. CONCLUSIONS: These findings demonstrate the important role of ASPM in the tumorigenesis and progression of LUAD.

In Situ Phase Transition of Elastin-Like Polypeptide Chains Regulates Thermoresponsive Properties of Elastomeric Protein-Based Hydrogels.

Engineering protein-based hydrogels that can change their physical and mechanical properties in response to environmental stimuli have attracted considerable interest due to their promising applications in biomedical engineering. Among environmental stimuli, temperature is of particular interest. Most thermally responsive protein hydrogels are constructed from thermally responsive elastin-like polypeptides (ELPs), which exhibit a lower critical solution temperature (LCST) transition, or nonstructured elastomeric proteins fused with ELPs. Here we report the engineering of thermally responsive elastomeric protein-based hydrogels by fusing ELPs to elastomeric proteins made of tandemly arranged folded globular proteins. By fusing ELP sequence (VPGVG)n to an elastomeric protein (GR)4, which is made of small globular protein GB1 (G) and random coil sequence resilin (R), we engineered a series of protein block copolymers, Vn-(GR)4. The fusion proteins Vn-(GR)4 exhibit temperature-responsive behaviors in aqueous solution that are different from that of Vn-ELPs, as they did not exhibit the macroscopic phase transitions in the turbidity test. Instead, V48-(GR)4 and V72-(GR)4 form micelles at temperatures higher than the transition temperature of V48 and V72 at the same concentration. Using the well-developed ruthenium-mediated photochemical cross-linking method, Vn-(GR)4 polymers can be cross-linked into hydrogels, in which Vn-ELP serve as side chains of the hydrogel network. These hydrogels exhibited thermoresponsive properties due to the temperature dependent phase transition behaviors of the incorporated Vn-ELPs blocks. At elevated temperatures, the Vn-ELPs side chains in the hydrogel network underwent aggregation, leading to secondary physical cross-linking. The aggregation of the Vn-ELPs resulted in higher Young's modulus and reduced swelling ratio. Furthermore, the amplitude of such property changes can be tuned by side chain length and composition. These results demonstrate that in situ phase behaviors of ELP side chains can regulate thermoresponsiveness of protein-based hydrogels. We anticipate that this method can be applied to other elastomeric proteins for potential biomedical applications.

An Injectable Self-Healing Protein Hydrogel with Multiple Dissipation Modes and Tunable Dynamic Response.

Hydrogels with dynamic mechanical properties are of special interest in the field of tissue engineering and drug delivery. However, it remains challenging to tailor the dynamic mechanical response of hydrogels to simultaneously meet diverse application needs. Here, we report a hetero-coiled-coil complex cross-linked protein hydrogel exhibiting unusual multiple energy dissipation modes and tunable dynamic response. Such unique features confer on the hydrogel responsiveness to mechanical stimuli in a broad range of frequencies. Therefore, the hydrogels are injectable due to their shearing-thinning properties at low shear rates of 0.8 rad s-1 and can fully recover their mechanical properties within a few seconds due to the intrinsic fast dynamics of the cross-linkers. Moreover, the dynamic response of these hydrogels can be fine-tuned by the temperature and the hydrogel network structures. We anticipate that these hydrogels are promising candidates for delivering therapeutic drugs, biological molecules, and cells in a broad spectrum of biomedical applications.

Engineering Protein-Clay Nanosheets Composite Hydrogels with Designed Arginine-Rich Proteins.

Clay nanosheets (CNSs) have been widely used in the design of nanocomposite biomaterials. CNSs display a disk-like morphology with strong negatively charged surfaces. It has been shown that guanidinium-containing molecules can bind CNSs through noncovalent salt-bridge interactions and thus serve as "molecular glues" for CNSs. Making use of the guanidinium side chain in arginine, here, we designed novel arginine-rich elastomeric proteins to engineer protein-CNS nanocomposite hydrogels. Our results showed that these arginine-rich proteins can interact with CNSs effectively and can cross-link CNSs into hydrogels. Rheological measurements showed that mechanical properties of the resultant hydrogels depended on the arginine content in the arginine-rich proteins as well as CNS/protein concentration. Compared with hydrogels constructed from CNSs or proteins alone, the novel protein-CNS nanocomposite hydrogels show much improved mechanical properties. Our work opens up a new avenue to engineer functional protein hydrogels for various applications.

Optically controlled reversible protein hydrogels based on photoswitchable fluorescent protein Dronpa.

Exploiting the optically controlled association and dissociation behavior of a photoswitchable fluorescent protein, Dronpa145N, here we demonstrate the engineering of an optically switchable reversible protein hydrogel using Dronpa145N-based protein building blocks. Our results open the possibility to optically tune the mechanical, chemical and structural properties of protein hydrogels.