Synthetic peptides for the precise transportation of proteins of interests to selectable subcellular areas.

Proteins, as gifts from nature, provide structure, sequence, and function templates for designing biomaterials. As first reported here, one group of proteins called reflectins and derived peptides were found to present distinct intracellular distribution preferences. Taking their conserved motifs and flexible linkers as Lego bricks, a series of reflectin-derivates were designed and expressed in cells. The selective intracellular localization property leaned on an RMs (canonical conserved reflectin motifs)-replication-determined manner, suggesting that these linkers and motifs were constructional fragments and ready-to-use building blocks for synthetic design and construction. A precise spatiotemporal application demo was constructed in the work by integrating RLNto2 (as one representative of a synthetic peptide derived from RfA1) into the Tet-on system to effectively transport cargo peptides into nuclei at selective time points. Further, the intracellular localization of RfA1 derivatives was spatiotemporally controllable with a CRY2/CIB1 system. At last, the functional homogeneities of either motifs or linkers were verified, which made them standardized building blocks for synthetic biology. In summary, the work provides a modularized, orthotropic, and well-characterized synthetic-peptide warehouse for precisely regulating the nucleocytoplasmic localization of proteins.

A Mini-Review on Reflectins, from Biochemical Properties to Bio-Inspired Applications.

Some cephalopods (squids, octopuses, and cuttlefishes) produce dynamic structural colors, for camouflage or communication. The key to this remarkable capability is one group of specialized cells called iridocytes, which contain aligned membrane-enclosed platelets of high-reflective reflectins and work as intracellular Bragg reflectors. These reflectins have unusual amino acid compositions and sequential properties, which endows them with functional characteristics: an extremely high reflective index among natural proteins and the ability to answer various environmental stimuli. Based on their unique material composition and responsive self-organization properties, the material community has developed an impressive array of reflectin- or iridocyte-inspired optical systems with distinct tunable reflectance according to a series of internal and external factors. More recently, scientists have made creative attempts to engineer mammalian cells to explore the function potentials of reflectin proteins as well as their working mechanism in the cellular environment. Progress in wide scientific areas (biophysics, genomics, gene editing, etc.) brings in new opportunities to better understand reflectins and new approaches to fully utilize them. The work introduced the composition features, biochemical properties, the latest developments, future considerations of reflectins, and their inspiration applications to give newcomers a comprehensive understanding and mutually exchanged knowledge from different communities (e.g., biology and material).

An essential role of disulfide bonds for the hierarchical self-assembly and underwater affinity of CP20-derived peptides.

Barnacles are typical fouling organisms strongly adhere to immersed solid substrates by secreting proteinaceous adhesives called cement proteins (CPs). The self-assembly of the CPs forms a permanently bonded layer that binds barnacles to foreign surfaces. However, it is difficult to determine their natural structure and describe their self-assembly properties due to the abundance of cysteines in whole-length CP20. A putative functional motif of Balanus albicostatus CP20 (BalCP20) was identified to present distinctive self-assembly and wet-binding characteristics. Atomic-force microscopy (AFM) and transmission electron microscope (TEM) investigations showed that wildtype BalCP20-P3 formed grain-like spindles, which assembled into fractal-like structures like ears of wheat. SDS-PAGE, AFM, and LSCM showed that DTT treatment opened up disulfide bonds between cysteines and disrupted fractal-like structures. Additionally, these morphologies were abolished when one of the BalCP20-P3 four cysteines was mutated by alanine. Circular dichroism (CD) results suggested that the morphological diversity among BalCP20-P3 and its mutations was related to the proportion of α-helices. Finally, quartz crystal microbalance with dissipation (QCM-D) detected that BalCP20-P3 and its mutations with diverse self-assemblies occupied different affinities. The above results demonstrated that cysteines and disulfide bonds played a crucial role in the self-assembly and wet binding of BalCP20-P3. The work provides new ideas for the underwater bonding of BalCP20 and developing new bionic underwater adhesives.

Tunable Cellular Localization and Extensive Cytoskeleton-Interplay of Reflectins.

Reflectin proteins are natural copolymers consisting of repeated canonical domains. They are located in a biophotonic system called Bragg lamellae and manipulate the dynamic structural coloration of iridocytes. Their biological functions are intriguing, but the underlying mechanism is not fully understood. Reflectin A1, A2, B1, and C were found to present distinguished cyto-/nucleoplasmic localization preferences in the work. Comparable intracellular localization was reproduced by truncated reflectin variants, suggesting a conceivable evolutionary order among reflectin proteins. The size-dependent access of reflectin variants into the nucleus demonstrated a potential model of how reflectins get into Bragg lamellae. Moreover, RfA1 was found to extensively interact with the cytoskeleton, including its binding to actin and enrichment at the microtubule organizing center. This implied that the cytoskeleton system plays a fundamental role during the organization and transportation of reflectin proteins. The findings presented here provide evidence to get an in-depth insight into the evolutionary processes and working mechanisms of reflectins, as well as novel molecular tools to achieve tunable intracellular transportation.

Adhesive Materials Inspired by Barnacle Underwater Adhesion: Biological Principles and Biomimetic Designs.

Wet adhesion technology has potential applications in various fields, especially in the biomedical field, yet it has not been completely mastered by humans. Many aquatic organisms (e.g., mussels, sandcastle worms, and barnacles) have evolved into wet adhesion specialists with excellent underwater adhesion abilities, and mimicking their adhesion principles to engineer artificial adhesive materials offers an important avenue to address the wet adhesion issue. The crustacean barnacle secretes a proteinaceous adhesive called barnacle cement, with which they firmly attach their bodies to almost any substrate underwater. Owing to the unique chemical composition, structural property, and adhesion mechanism, barnacle cement has attracted widespread research interest as a novel model for designing biomimetic adhesive materials, with significant progress being made. To further boost the development of barnacle cement-inspired adhesive materials (BCIAMs), it is necessary to systematically summarize their design strategies and research advances. However, no relevant reviews have been published yet. In this context, we presented a systematic review for the first time. First, we introduced the underwater adhesion principles of natural barnacle cement, which lay the basis for the design of BCIAMs. Subsequently, we classified the BCIAMs into three major categories according to the different design strategies and summarized their research advances in great detail. Finally, we discussed the research challenge and future trends of this field. We believe that this review can not only improve our understanding of the molecular mechanism of barnacle underwater adhesion but also accelerate the development of barnacle-inspired wet adhesion technology.

Short Peptides Derived from a Block Copolymer-like Barnacle Cement Protein Self-Assembled into Diverse Supramolecular Structures.

Peptides capable of self-assembling into different supramolecular structures have potential applications in a variety of areas. The biomimetic molecular design offers an important avenue to discover novel self-assembling peptides. Despite this, a lot of biomimetic self-assembling peptides have been reported so far; to continually expand the scope of peptide self-assembly, it is necessary to find out more novel self-assembling peptides. Barnacle cp19k, a key underwater adhesive protein, shows special block copolymer-like characteristics and diversified self-assembly properties, providing an ideal template for biomimetic peptide design. In this study, inspired by Balanus albicostatus cp19k (Balcp19k), we rationally designed nine biomimetic peptides (P1-P9) and systematically studied their self-assembly behaviors for the first time. Combining microscale morphology observations and secondary structure analyses, we found that multiple biomimetic peptides derived from the central region and the C-terminus of Balcp19k form distinct supramolecular structures via different self-assembly mechanisms under acidic conditions. Specifically, P9 self-assembles into typical amyloid fibers. P7, which resembles ionic self-complementary peptides by containing nonstrictly alternating hydrophobic and charged amino acids, self-assembles into uniform, discrete nanofibers. P6 with amphipathic features forms twisted nanoribbons. Most interestingly, P4 self-assembles to form helical nanofibers and novel ring-shaped microstructures, showing unique self-assembly behaviors. Apart from their self-assembly properties, these peptides showed good cytocompatibility and demonstrated promising applications in biomedical areas. Our results expanded the repertoire of self-assembling peptides and provided new insights into the structure-function relationship of barnacle cp19k.

Rose Pistil Stigma: Hierarchical Superhydrophobic Surfaces with Hydrophilic Microtips for Microdroplet Manipulation.

Finger-like radial hierarchical micropillars with folded tips are observed on the surface of the rose pistil stigma (RPS). Impressively, a water droplet on the surface of the RPS presents a spherical shape and it still hangs on the surface even when the RPS is turned over. Superhydrophobicity and high adhesion to water are demonstrated on the RPS, which is beneficial for the RPS to remain clean and fresh. The special wetting behavior of the RPS is highly related to its hierarchical microstructures and surface chemistry. Finger-like hierarchical micropillars with a high aspect ratio are capable of retaining air to support superhydrophobicity while the microgap between the micropillars and on the hydrophilic tips enables the RPS to retain a high adhesion to water. These findings about the unique wetting behaviors of the RPS may provide inspiration for the design and fabrication of functional wetting surfaces for diverse applications such as microdroplet manipulation, three-dimensional cell culture, and microfluidics.

Transcriptome analyses reveal the utilization of nitrogen sources and related metabolic mechanisms of Sporosarcina pasteurii.

In recent years, Sporosarcina pasteurii (S. pasteurii) has become one of the most popular bacteria in microbially induced calcium carbonate precipitation (MICP). Various applications have been developed based on the efficient urease that can induce the precipitation of calcium carbonate. However, the metabolic mechanism related to biomineralization of S. pasteurii has not been clearly elucidated. The process of bacterial culture and biomineralization consumes a large amount of urea or ammonium salts, which are usually used as agricultural fertilizers, not to mention probable environmental pollutions caused by the excessive use of these raw materials. Therefore, it is urgent to reveal the mechanism of nitrogen utilization and metabolism of S. pasteurii. In this paper, we compared the growth and gene expression of S. pasteurii under three different culture conditions through transcriptome analyses. GO and KEGG analyses revealed that both ammonium and urea were direct nitrogen sources of S. pasteurii, and the bacteria could not grow normally in the absence of ammonium or urea. To the best of our knowledge, this paper is the first one to reveal the nitrogen utilization mechanism of S. pasteurii through transcriptome methods. Furthermore, the presence of ammonium might promote the synthesis of intracellular ATP and enhance the motility of the bacteria. There should be an ATP synthesis mechanism associated with urea hydrolysis catalyzed by urease in S. pasteurii.

Transcriptome analyses suggest a molecular mechanism for the SIPC response of Amphibalanus amphitrite.

Barnacles are notorious marine fouling organisms. Their successful attachment to a substrate requires that they search for an appropriate habitat during their cyprid stage. A chemical cue called SIPC (Settlement-Inducing Protein Complex) has been shown to play a key role in the induction of cyprid gregarious settlement; however, the underlying biochemical mechanism remains unclear. Here, RNA-seq was used to examine the gene expression profiles of Amphibalanus amphitrite cyprids in response to SIPC and to identify SIPC-activated intracellular signaling pathways. A total of 389 unigenes were differentially expressed in response to SIPC, and cement protein genes were not among them. KEGG enrichment analysis suggested that SNARE interactions in the vesicular transport pathway were significantly influenced by SIPC treatment, indicating a possible role for SIPC in triggering protein transportation and secretion. Several genes with specific functions in metamorphosis were found among the differentially expressed genes (DEGs). GO (Gene Ontology) enrichment analysis revealed that the DEGs were significantly enriched in enamel mineralization pathways, suggesting that SIPC may also be involved in the activation of mineralization.

Bioinspired Multiscale Wrinkling Patterns on Curved Substrates: An Overview.

The surface wrinkling of biological tissues is ubiquitous in nature. Accumulating evidence suggests that the mechanical force plays a significant role in shaping the biological morphologies. Controlled wrinkling has been demonstrated to be able to spontaneously form rich multiscale patterns, on either planar or curved surfaces. The surface wrinkling on planar substrates has been investigated thoroughly during the past decades. However, most wrinkling morphologies in nature are based on the curved biological surfaces and the research of controllable patterning on curved substrates still remains weak. The study of wrinkling on curved substrates is critical for understanding the biological growth, developing three-dimensional (3D) or four-dimensional (4D) fabrication techniques, and creating novel topographic patterns. In this review, fundamental wrinkling mechanics and recent advances in both fabrications and applications of the wrinkling patterns on curved substrates are summarized. The mechanics behind the wrinkles is compared between the planar and the curved cases. Beyond the film thickness, modulus ratio, and mismatch strain, the substrate curvature is one more significant parameter controlling the surface wrinkling. Curved substrates can be both solid and hollow with various 3D geometries across multiple length scales. Up to date, the wrinkling morphologies on solid/hollow core-shell spheres and cylinders have been simulated and selectively produced. Emerging applications of the curved topographic patterns have been found in smart wetting surfaces, cell culture interfaces, healthcare materials, and actuators, which may accelerate the development of artificial organs, stimuli-responsive devices, and micro/nano fabrications with higher dimensions.

Self-Assembled Nanofibers for Strong Underwater Adhesion: The Trick of Barnacles.

Developing adhesives that can function underwater remains a major challenge for bioengineering, yet many marine creatures, exemplified as mussels and barnacles, have evolved their unique proteinaceous adhesives for strong wet adhesion. The mechanisms underlying the strong adhesion of these natural adhesive proteins provide rich information for biomimetic efforts. Here, combining atomic force microscopy (AFM) imaging and force spectroscopy, we examine the effects of pH on the self-assembly and adhesive properties of cp19k, a key barnacle underwater adhesive protein. For the first time, we confirm that the bacterial recombinant Balanus albicostatus cp19k (rBalcp19k), which contains no 3,4-dihydroxyphenylalanine (DOPA) or any other amino acids with post-translational modifications, can self-assemble into aggregated nanofibers at acidic pHs. Under moderately acidic conditions, the adhesion strength of unassembled monomeric rBalcp19k on mica is only slightly lower than that of a commercially available mussel adhesive protein mixture, yet the adhesion ability of rBalcp19k monomers decreases significantly at increased pH. In contrast, upon preassembly at acidic and low-salinity conditions, rBalcp19k nanofibers keep stable in basic and high-salinity seawater and display much stronger adhesion and thus show resistance to its adverse impacts. Besides, we find that the adhesion ability of Balcp19k is not impaired when it is combined with an N-terminal Thioredoxin (Trx) tag, yet whether the self-assembly property will be disrupted is not determined. Collectively, the self-assembly-enhanced adhesion presents a previously unexplored mechanism for the strong wet adhesion of barnacle cement proteins and may lead to the design of barnacle-inspired adhesive materials.

Amyloid fibril aggregation: An insight into the underwater adhesion of barnacle cement.

Barnacles robustly adhere themselves to diverse submarine substrates through a proteinaceous complex termed the "barnacle cement". Previous studies have indicated that certain peptides derived from some barnacle cement proteins can self-assemble into amyloid fibrils. In this study, we assessed the self-assembly behavior of a full-length 19 kDa cement protein from Balanus albicostatus (Balcp19k) in different buffers. Results of Thioflavin T binding assay, transmission electron microscopy, and Fourier transform infrared spectroscopy suggested that the bacterial recombinant Balcp19k was able to aggregate into typical amyloid fibrils. The time required for the self-assembly process was close to that required for the complete curing of barnacle cement complex. Moreover, the solubility of Balcp19k amyloid deposits in guanidine hydrochloride and urea was same as that of the cured cement. These results indicated the inherent self-assembling nature of Balcp19k, implying that the amyloid fibril formation plays a critical role in barnacle cement curing procedure and its insolubility. Our results should be conducive to understanding barnacle underwater adhesion mechanisms and have implications in the development of new-generation antifouling techniques and in the designing of novel wet adhesives for biomedical and technical applications.

Photoreceptor PhyB Involved in Arabidopsis Temperature Perception and Heat-Tolerance Formation.

The influence of temperature on plants is essential. However, our knowledge on the intricate regulation process underlying heat stress (HS) response in plants is limited. Recently, information about thermal sensors in vivo has begun to emerge. In this study, another primary environmental stimulus, light, was verified once again to work with temperature synergistically on plants, through the modulation of numerous biological processes. With the application of transcriptomic analysis, a substantial number of heat-responsive genes were detected involved in both light- and phytohormone-mediated pathways in Arabidopsis. During this process, phytoreceptor phyB acts as a molecular switch to turn on or turn off several other genes HS response, under different light conditions. Furthermore, a morphological study showed the afunction of phyB enhanced plants thermal tolerance, confirming the important role of this phytochrome in temperature perception and response in plants. This study adds data to the picture of light and temperature signaling cross-talk in plants, which is important for the exploration of complicated HS responses or light-mediated mechanisms. Furthermore, based on its influence on Arabidopsis thermal response in both morphological and physiological levels, phyB is a photoreceptor, as revealed before, as well as an essential thermal sensor in plants.

Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+) Plasmid Having Adhesion Strength Comparable to Several Commercial Glues.

The barnacle is well known for its tenacious and permanent attachment to a wide variety of underwater substrates, which is accomplished by synthesizing, secreting and curing a mixture of adhesive proteins termed "barnacle cement". In order to evaluate interfacial adhesion abilities of barnacle cement proteins, the cp19k homologous gene in Balanus albicostatus (Balcp19k) was cloned and expressed in Escherichia coli. Here, we report an intriguing discovery of a gel-like super adhesive aggregation produced by Trx-Balcp19k, a recombinant Balcp19k fusion protein. The Trx-Balcp19k consists of an 18 kDa fragment at the N-terminus, which is encoded by pET-32a(+) plasmid and mainly comprised of a thioredoxin (Trx) tag, and Balcp19k at the C-terminus. The sticky aggregation was designated as "Trx-Balcp19k gel", and the bulk adhesion strength, biochemical composition, as well as formation conditions were all carefully investigated. The Trx-Balcp19k gel exhibited strong adhesion strength of 2.10 ± 0.67 MPa, which was approximately fifty folds higher than that of the disaggregated Trx-Balcp19k (40 ± 8 kPa) and rivaled those of commercial polyvinyl acetate (PVA) craft glue (Mont Marte, Australia) and UHU glue (UHU GmbH & Co. KG, Germany). Lipids were absent from the Trx-Balcp19k gel and only a trace amount of carbohydrates was detected. We postulate that the electrostatic interactions play a key role in the formation of Trx-Balcp19k gel, by mediating self-aggregation of Trx-Balcp19k based on its asymmetric distribution pattern of charged amino acids. Taken together, we believe that our discovery not only presents a promising biological adhesive with potential applications in both biomedical and technical fields, but also provides valuable paradigms for molecular design of bio-inspired peptide- or protein-based materials.

Chitosan coated on the layers' glucose oxidase immobilized on cysteamine/Au electrode for use as glucose biosensor.

A glucose biosensor was developed via direct immobilization of glucose oxidase (GOD) by self-assembled cysteamine monolayer on Au electrode surface followed by coating chitosan on the surface of electrode. In this work, chitosan film was coated on the surface of GOD as a protection film to ensure the stability and biocompatibility of the constructed glucose biosensor. The different application ranges of sensors were fabricated by immobilizing varied layers of GOD. The modified surface film was characterized by a scanning electron microscope (SEM) and the fabrication process of the biosensor was confirmed through electrochemical impedance spectroscopy (EIS) of ferrocyanide. The performance of cyclic voltammetry (CV) in the absence and presence of 25 mM glucose and ferrocenemethanol showed a diffusion-controlled electrode process and reflected the different maximum currents between the different GOD layers. With the developed glucose biosensor, the detection limits of the two linear responses are 49.96 µM and 316.8 µM with the sensitivities of 8.91 µA mM(-1)cm(-2) and 2.93 µA mM(-1)cm(-2), respectively. In addition, good stability (up to 30 days) of the developed biosensor was observed. The advantages of this new method for sensors construction was convenient and different width ranges of detection can be obtained by modified varied layers of GOD. The sensor with two layers of enzyme displayed two current linear responses of glucose. The present work provided a simplicity and novelty method for producing biosensors, which may help design enzyme reactors and biosensors in the future.

[Design of plant leaf bionic camouflage materials based on spectral analysis].

The influence of structure parameters and contents of plant leaves on their reflectance spectra was analyzed using the PROSPECT model. The result showed that the bionic camouflage materials should be provided with coarse surface and spongy inner structure, the refractive index of main content must be close to that of plant leaves, the contents of materials should contain chlorophyll and water, and the content of C-H bond must be strictly controlled. Based on the analysis above, a novel camouflage material, which was constituted by coarse transparent waterproof surface, chlorophyll, water and spongy material, was designed. The result of verifiable experiment showed that the reflectance spectra of camouflage material exhibited the same characteristics as those of plant leaves. The similarity coefficient of reflectance spectrum of the camouflage material and camphor leaves was 0.988 1, and the characteristics of camouflage material did not change after sunlight treatment for three months. The bionic camouflage material, who exhibited a high spectral similarity with plant leaves and a good weather resistance, will be an available method for reconnaissance of hyperspectral imaging hopefully.

Fabrication of functional bioinorganic nanoconstructs by polymer-silica wrapping of individual myoglobin molecules.

Discrete core-shell hybrid nanoparticles comprising individual met-myoglobin (met-Mb) molecules incarcerated within an ultrathin polymer/silica shell were prepared without loss of biofunctionality by a facile self-assembly procedure. Solubilisation of met-Mb in cyclohexane in the near-absence of water was achieved by wrapping individual protein molecules in the amphiphilic triblock copolymer poly(ethylene-oxide)19-poly(propylene-oxide)69-poly(ethylene-oxide)19 (EO19-PO69-EO19, P123). Addition of tetramethoxysilane to the met-Mb/P123 conjugates in cyclohexane produced discrete nanoparticles that contained protein, polymer and silica, and which were 3-5.5 nm in size, consistent with the entrapment of single molecules of met-Mb. The hybrid nanoconstructs were isolated and re-dispersed in water without loss of secondary structure, and remained functionally active with respect to redox reactions and CO and O2 ligand binding at the porphyrin metallocentre. The incarcerated met-Mb biomolecules showed enhanced thermal stability up to a temperature of around 85 °C. These properties, along with the high biocompatibility of silica and P123, suggest that the silicified protein-polymer constructs could be utilised as functional nanoscale components in bionanotechnology.