bHLH transcription factors Hes1, Ascl1 and Oligo2 exhibit different expression patterns in the process of physiological electric fields-induced neuronal differentiation.

BACKGROUND: Recent studies have shown that the expression of bHLH transcription factors Hes1, Ascl1, and Oligo2 has an oscillating balance in neural stem cells (NSCs) to maintain their self-proliferation and multi-directional differentiation potential. This balance can be disrupted by exogenous stimulation. Our previous work has identified that electrical stimulation could induce neuronal differentiation of mouse NSCs. METHODS: To further evaluate if physiological electric fields (EFs)-induced neuronal differentiation is related to the expression patterns of bHLH transcription factors Hes1, Ascl1, and Oligo2, mouse embryonic brain NSCs were used to investigate the expression changes of Ascl1, Hes1 and Oligo2 in mRNA and protein levels during EF-induced neuronal differentiation. RESULTS: Our results showed that NSCs expressed high level of Hes1, while expression of Ascl1 and Oligo2 stayed at very low levels. When NSCs exited proliferation, the expression of Hes1 in differentiated cells began to decrease and oscillated at the low expression level. Oligo2 showed irregular changes in low expression level. EF-stimulation significantly increased the expression of Ascl1 at mRNA and protein levels accompanied by an increased percentage of neuronal differentiation. What's more, over-expression of Hes1 inhibited the neuronal differentiation induced by EFs. CONCLUSION: EF-stimulation directed neuronal differentiation of NSCs by promoting the continuous accumulation of Ascl1 expression and decreasing the expression of Hes1.

A review of the evidence to support electrical stimulation-induced vascularization in engineered tissue.

Tissue engineering presents a promising solution for regenerative medicine and the success depends on the supply of oxygen/nutrients to the cells by rapid vascularization. More and more technologies are being developed to facilitate vascularization of engineered tissues. In this review, we indicated that a regulatory system which influences all angiogenesis associated cells to achieve their desired functional state is ideal for the construction of vascularized engineered tissues in vitro. We presented the evidence that electrical stimulation (ES) enhances the synergistic promotion of co-cultured angiogenesis associated cells and its potential regulatory mechanisms, highlighted the potential advantages of a combination of mesenchymal stem cells (MSCs), endothelial cells (ECs) and ES to achieve tissue vascularization, with particular emphasis on the different biological pathways of ES-regulated ECs. Finally, we proposed the future direction of using ES to reconstruct engineered tissue blood vessels, pointed out the potential advantages and disadvantages of ES application on tissue vascularization.

Electrical stimulation induced structural 3D human engineered neural tissue with well-developed neuronal network and functional connectivity.

Objective.Three-dimensional (3D) neural tissue engineering is expected to provide new stride in developing neural disease models and functional substitutes to aid in the treatment of central nervous system injury. We have previously detailed an electrical stimulation (ES) system to generate 3D mouse engineered neural tissue (mENT)in vitro. However, ES-induced human ENT (hENT) has not previously been either investigated or identified in structural and functional manner. Here, we applied ES as a stimulator to regulate human neural stem cells in 3D Matrigel, explored the components and functional properties of hENTs.Approach.By immunofluorescence chemical staining and electron microscope imaging, we evaluated the effects of ES on (1) neuronal differentiation and maturation, (2) neurites outgrowth and alignment in hENT, (3) formation of synapses and myelin sheaths in hENT. We further investigated the formation of synaptic connections betweenex-vivo-fused mouse and human tissue. We used calcium imaging to detect activities of neurons in hENT culture.Results.ES could induce neuronal differentiation, the orderly growth of neurites and the maturation of neuron subtypes to construct a well-developed neuronal network with synapses and myelin sheaths. Most importantly, we discovered that raising extracellular K+concentration resulted the increasing neuronal excitability in the hENT, indicating electrical activities in neuronal cells.Significance.We applied ES to generate the organised 3D hENTs and identified them in both structural and functional manner.

Selection and evolution of disulfide-rich peptides via cellular protein quality control.

Disulfide-rich peptides (DRPs) are an interesting and promising molecular format for drug discovery and development. However, the engineering and application of DRPs rely on the foldability of the peptides into specific structures with correct disulfide pairing, which strongly hinders the development of designed DRPs with randomly encoded sequences. Design or discovery of new DRPs with robust foldability would provide valuable scaffolds for developing peptide-based probes or therapeutics. Herein we report a cell-based selection system leveraging cellular protein quality control (termed PQC-select) to select DRPs with robust foldability from random sequences. By correlating the foldability of DRPs with their expression levels on the cell surface, thousands of sequences that can fold properly have been successfully identified. We anticipated that PQC-select will be applicable to many other designed DRP scaffolds in which the disulfide frameworks and/or the disulfide-directing motifs can be varied, enabling the generation of a variety of foldable DRPs with new structures and superior potential for further developments.

Effects of Electrical Stimulation on Articular Cartilage Regeneration with a Focus on Piezoelectric Biomaterials for Articular Cartilage Tissue Repair and Engineering.

There is increasing evidence that chondrocytes within articular cartilage are affected by endogenous force-related electrical potentials. Furthermore, electrical stimulation (ES) promotes the proliferation of chondrocytes and the synthesis of extracellular matrix (ECM) molecules, which accelerate the healing of cartilage defects. These findings suggest the potential application of ES in cartilage repair. In this review, we summarize the pathogenesis of articular cartilage injuries and the current clinical strategies for the treatment of articular cartilage injuries. We then focus on the application of ES in the repair of articular cartilage in vivo. The ES-induced chondrogenic differentiation of mesenchymal stem cells (MSCs) and its potential regulatory mechanism are discussed in detail. In addition, we discuss the potential of applying piezoelectric materials in the process of constructing engineering articular cartilage, highlighting the important advances in the unique field of tissue engineering.

From 2D to 3D Co-Culture Systems: A Review of Co-Culture Models to Study the Neural Cells Interaction.

The central nervous system (CNS) controls and regulates the functional activities of the organ systems and maintains the unity between the body and the external environment. The advent of co-culture systems has made it possible to elucidate the interactions between neural cells in vitro and to reproduce complex neural circuits. Here, we classified the co-culture system as a two-dimensional (2D) co-culture system, a cell-based three-dimensional (3D) co-culture system, a tissue slice-based 3D co-culture system, an organoid-based 3D co-culture system, and a microfluidic platform-based 3D co-culture system. We provide an overview of these different co-culture models and their applications in the study of neural cell interaction. The application of co-culture systems in virus-infected CNS disease models is also discussed here. Finally, the direction of the co-culture system in future research is prospected.

Structure-guided design of CPPC-paired disulfide-rich peptide libraries for ligand and drug discovery.

Peptides constrained through multiple disulfides (or disulfide-rich peptides, DRPs) have been an emerging frontier for ligand and drug discovery. Such peptides have the potential to combine the binding capability of biologics with the stability and bioavailability of smaller molecules. However, DRPs with stable three-dimensional (3D) structures are usually of natural origin or engineered from natural ones. Here, we report the discovery and identification of CPPC (cysteine-proline-proline-cysteine) motif-directed DRPs with stable 3D structures (i.e., CPPC-DRPs). A range of new CPPC-DRPs were designed or selected from either random or structure-convergent peptide libraries. Thus, for the first time we revealed that the CPPC-DRPs can maintain diverse 3D structures by taking advantage of constraints from unique dimeric CPPC mini-loops, including irregular structures and regular α-helix and ß-sheet folds. New CPPC-DRPs that can specifically bind the receptors (CD28) on the cell surface were also successfully discovered and identified using our DRP-discovery platform. Overall, this study provides the basis for accessing an unconventional peptide structure space previously inaccessible by natural DRPs and computational designs, inspiring the development of new peptide ligands and therapeutics.

Physiological Electric Field: A Potential Construction Regulator of Human Brain Organoids.

Brain organoids can reproduce the regional three-dimensional (3D) tissue structure of human brains, following the in vivo developmental trajectory at the cellular level; therefore, they are considered to present one of the best brain simulation model systems. By briefly summarizing the latest research concerning brain organoid construction methods, the basic principles, and challenges, this review intends to identify the potential role of the physiological electric field (EF) in the construction of brain organoids because of its important regulatory function in neurogenesis. EFs could initiate neural tissue formation, inducing the neuronal differentiation of NSCs, both of which capabilities make it an important element of the in vitro construction of brain organoids. More importantly, by adjusting the stimulation protocol and special/temporal distributions of EFs, neural organoids might be created following a predesigned 3D framework, particularly a specific neural network, because this promotes the orderly growth of neural processes, coordinate neuronal migration and maturation, and stimulate synapse and myelin sheath formation. Thus, the application of EF for constructing brain organoids in a3D matrix could be a promising future direction in neural tissue engineering.

De novo design and directed folding of disulfide-bridged peptide heterodimers.

Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Computational methods have also been developed to design heterodimers of advanced functions. However, these peptide heterodimers are usually formed through noncovalent interactions, which are prone to dissociate and subject to concentration-dependent nonspecific aggregation. Heterodimers crosslinked with interchain disulfide bonds are more stable, but it represents a formidable challenge for both the computational design of heterodimers and the manipulation of disulfide pairing for heterodimer synthesis and applications. Here, we report the design, synthesis and application of interchain disulfide-bridged peptide heterodimers with mutual orthogonality by combining computational de novo designs with a directed disulfide pairing strategy. These heterodimers can be used as not only scaffolds for generating functional molecules but chemical tools or building blocks for protein labeling and construction of crosslinking hybrids. This study thus opens the door for using this unexplored dimeric structure space for many biological applications.

Inhibition of invadopodia formation by diosgenin in tumor cells.

Diosgenin is a type of steroid extracted from the rhizome of Dioscorea plants. In traditional Chinese medicine, Dioscorea has the effect of 'eliminating phlegm, promoting digestion, relaxing tendons, promoting blood circulation and inhibiting malaria'. Recent studies have confirmed that diosgenin exhibits a number of pharmacological effects, including antitumor activities. Through its antitumor effect, diosgenin is able to block tumor progression and increase the survival rate of patients with cancer; ultimately improving their quality of life. However, the mechanism underlying its pharmacological action remains unclear. Once tumor cells reach a metastatic phase, it can be fatal. Increased migration and invasiveness are the hallmarks of metastatic tumor cells. Invadopodia formation is key to maintaining the high migration and invasive ability of tumor cells. Invadopodia are a type of membrane structure process rich in filamentous-actin and are common in highly invasive tumor cells. In addition to actin, numerous actin regulators, including cortical actin-binding protein (Cortactin), accumulate in invadopodia. Cortactin is a microfilament actin-binding protein with special repetitive domains that are directly involved in the formation of the cortical microfilament actin cell skeleton. Cortactin is also one of the main substrates of intracellular Src-type tyrosine protein kinases and represents a highly conserved family of intracellular cortical signaling proteins. In recent years, great progress has been made in understanding the role of Cortactin and its molecular mechanism in cell motility. However, the diosgenin-Cortactin-invadopodia mechanism is still under investigation. Therefore, the present review focused on the current research on the regulation of invadopodia by diosgenin via Cortactin.

Directed Disulfide Pairing and Folding of Peptides for the De Novo Development of Multicyclic Peptide Libraries.

Disulfide-rich peptides (DRPs) have been an emerging frontier for drug discovery. There have been two DRPs approved as drugs (i.e., Ziconotide and Linaclotide), and many others are undergoing preclinical studies or in clinical trials. All of these DRPs are of nature origin or derived from natural peptides. It is still a challenge to design new DRPs without recourse to natural scaffolds due to the difficulty in handling the disulfide pairing. Here we developed a simple and robust strategy for directing the disulfide pairing and folding of peptides with up to six cysteine residues. Our strategy exploits the dimeric pairing of CPPC (cysteine-proline-proline-cysteine) motifs for directing disulfide formation, and DRPs with different multicyclic topologies were designed and synthesized by regulating the patterns of CPPC motifs and cysteine residues in peptides. As neither sequence manipulations nor unnatural amino acids are involved, the designed DRPs can be used as templates for the de novo development of biosynthetic multicyclic peptide libraries, enabling selection of DRPs with new functions directly from fully randomized sequences. We believe that this work represents as an important step toward the discovery and design of new multicyclic peptide ligands and therapeutics with structures not derived from natural scaffolds.

Combination of electrical stimulation and bFGF synergistically promote neuronal differentiation of neural stem cells and neurite extension to construct 3D engineered neural tissue.

OBJECTIVE: The construction of in vitro three-dimensional (3D) neural tissue has to overcome two main types of challenges: (1) How to obtain enough number of functional neurons from stem cells in 3D culture; (2) How to wire those lately developed neurons into functional neural networks. Here, we describe the potential of using direct current (DC) electric field (EF) together with basic fibroblast growth factor (bFGF) synergistically in promoting neural stem cell (NSC) neuronal differentiation following by directing neurite outgrowth in the 3D neural tissue construction. APPROACH: By adjusting the electrical stimulation setup in this study, long-term electrical stimulation could be present in vitro. At an EF strength of 150 mV mm-1, cell responses, including cell viability, neuronal differentiation, cell morphology, the length of neuronal processes, synaptic structure and neural network formation, were quantified and analyzed. MAIN RESULTS: Analysis revealed that NSCs showed no significant cell death after certain EF treatments. EF-stimulated NSCs in 3D Matrigel mainly differentiated into neurons, but unlike NSCs in two-dimensional conditions, their processes were flat and stunted. When combined with bFGF, EF stimulation provided appropriate bioactive cues to establish engineered neural tissue with a proper neuronal cell number, highly branched neurites, and a well-developed neuronal network. SIGNIFICANCE: It is for the first time the synergistic effects of EF and bFGF stimulation have been evaluated in inducing the differentiation of NSCs into neurons and the acquisition of long neurites in a culture environment of in vitro 3D model. These optimized conditions may allow a well-developed neuronal network to be established within hydrogel droplets.

Condensation of 2-((Alkylthio)(aryl)methylene)malononitrile with 1,2-Aminothiol as a Novel Bioorthogonal Reaction for Site-Specific Protein Modification and Peptide Cyclization.

Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems. Herein we report that 1,2-aminothiol can react rapidly, specifically and efficiently with 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) under biocompatible conditions. This reaction undergoes a unique mechanism involving thiol-vinyl sulfide exchange, cyclization, and elimination of dicyanomethanide to form 2-aryl-4,5-dihydrothiazole (ADT) as a stable product. An 1,2-aminothiol functionality can be introduced into a peptide or a protein as an N-terminal cysteine or an unnatural amino acid. The bioorthogonality of this reaction was demonstrated by site-specific labeling of not only synthetic peptides and a purified recombinant protein but also proteins on mammalian cells and phages. Unlike other reagents in bioorthogonal reactions, the chemical and physical properties of TAMM can be easily tuned. TAMM can also be applied to generate phage-based ADT-cyclic peptide libraries without reducing phage infectivity. Using this approach, we identified ADT-cyclic peptides with high affinity to different protein targets, providing valuable tools for biological studies and potential therapeutics. Furthermore, the mild reaction conditions of TAMM condensation warrant its use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.

Stabilizing p-Dithiobenzyl Urethane Linkers without Rate-Limiting Self-Immolation for Traceless Drug Release.

Exploiting the redox sensitivity of disulfide bonds is a prevalent strategy in targeted prodrug designs. In contrast to aliphatic disulfides, p-thiobenzyl-based disulfides have rarely been used for prodrug designs, given their intrinsic instability caused by the low pKa of aromatic thiols. Here, we examined the interplay between steric hindrance and the low-pKa effect on thiol-disulfide exchange reactions and uncovered a new thiol-disulfide exchange process for the self-immolation of p-thiobenzyl-based disulfides. We observed a central leaving group shifting effect in the α,α-dimethyl-substituted p-dithiobenzyl urethane linkers (DMTB linkers), which leads to increased disulfide stability by more than two orders of magnitude, an extent that is significantly greater than that observed with typical aliphatic disulfides. In particular, the DMTB linkers display not only high stability, but also rapid self-immolation kinetics due to the low pKa of the aromatic thiol, which can be used as a general and robust linkage between targeting reagents and cytotoxic drugs for targeted prodrug designs. The unique and promising stability characteristics of the present DMTB linker will likely inspire the development of novel targeted prodrugs to achieve traceless release of drugs into cells.

Design and Synthesis of Cross-Link-Dense Peptides by Manipulating Regioselective Bisthioether Cross-Linking and Orthogonal Disulfide Pairing.

Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space because natural evolution and de novo design only keep sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with predefined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least one to three possible isomers. Here, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse cross-link-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).

Ascl1 Regulates Electric Field-Induced Neuronal Differentiation Through PI3K/Akt Pathway.

Directing differentiation of neural stem/progenitor cells (NSCs/NPCs) to produce functional neurons is one of the greatest challenges in regenerative medicine. Our previous paper has confirmed that electrical stimulation has a high efficiency of triggering neuronal differentiation by using isolated filum terminale (FT)-derived NPCs. To further clarify the intrinsic molecular mechanisms, protein-protein interaction (PPI) network analysis was applied to pinpoints novel hubs in electric field (EF)-induced neuronal differentiation. In this study, siRNA transfection of Achaete-scute homolog 1 (Ascl1) in NPCs or NPCs was followed by direct current stimulation at 150 mV/mm. Neuronal differentiation rate and protein expression level were analyzed after 7 or 14 days of electrical stimulation. The data showed that the expression level of Ascl1 was enhanced by electrical stimulation and positively correlated to EF strength. Moreover, we identified that the expression of Ascl1 positively regulated neuronal differentiation of NPCs and can be up-regulated by EF-stimulation through the activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway. Therefore, this study provides new insights into the role of Ascl1 and its relevant PI3K/Akt pathway in regulating of EF-induced neuronal differentiation and pointed out that continuous expression of Ascl1 in NPCs is required for EF-induced neuronal differentiation.

CXC-Mediated Cellular Uptake of Miniproteins: Forsaking "Arginine Magic".

Miniproteins have a size between that of larger biologics and small molecules and presumably possess the advantages of both; they represent an expanding class of promising scaffolds for the design of affinity reagents, enzymes, and therapeutics. Conventional strategies to promote cellular uptake of miniproteins rely on extensive grafting or embedding of arginine residues. However, the requirement of using cationic arginines would cause problems to the modified miniproteins, for example, low solubility in solutions (proneness of aggregation) and potential toxicity, which are open secrets in the peptide and protein communities. In this work, we report that the cell-permeability of cationic miniproteins can be further markedly increased through appending a magic CXC (cysteine- any-cysteine) motif, which takes advantage of thiol-disulfide exchanges on the cell surface. More importantly, we discovered that the high cell permeability of the CXC-appended miniproteins can still be preserved when the embedded arginines are all substituted with lysine residues, indicating that the "arginine magic" essential to almost all cell-permeable peptides and (mini)proteins is not required for the CXC-mediated cellular uptake. This finding provides a new avenue for designing highly cell-permeable miniproteins without compromise of potential toxicity and stability arising from arginine embedding or grafting.

De novo design of constrained and sequence-independent peptide scaffolds with topologically-formidable disulfide connectivities.

Disulfide-rich peptides are interesting scaffolds for drug design and discovery. However, peptide scaffolds constrained by disulfide bonds, either naturally occurring or computationally designed, have been suffering from the elusive (oxidative) folding behavior complying with Anfinsen's dogma, which strongly restricts their applicability in bioactive peptide design and discovery; because when primary peptide sequences are extensively manipulated, their disulfide connectivities might become scrambled. Here we present the design of cysteine/penicillamine (C/Pen)-mixed peptide frameworks that are capable of folding into specific regioisomers without dependence on primary amino acid sequences. Even certain folds that are considered to be topologically formidable can be generated in high yields. Currently, almost all disulfide-rich peptide scaffolds are vitally correlated to primary amino acid sequences, but ours are exceptional. These scaffolds should be of particular interest for further designing constrained peptides with new structures and functions, and more importantly, the ultimately designed peptides would not suffer from general oxidative folding problems.

Proteolytic Unlocking of Ultrastable Twin-Acylhydrazone Linkers for Lysosomal Acid-Triggered Release of Anticancer Drugs.

Targeted prodrugs exploiting cleavable linkers capable of responding to endogenous stimuli have increasingly been explored for cancer therapy. Successful application of these prodrug designs relies on the manipulation of both stability and responsiveness of the cleavable linkers, which, however, are difficult to be finely regulated, particularly for acid-responsive acylhydrazone bonds. Here we developed a new class of peptide-bridged twin-acylhydrazone linkers (PTA linkers) displaying both an ultrahigh stability and a rapid responsiveness-highly stable in neutral and acidic conditions due to the effect of cooperativity between the two acylhydrazone bonds, easily cleavable in acidic conditions after enzymatically triggered unlocking of the two bonds. Moreover, our study shows the design of PTA-linked prodrugs and the proof-of-concept application of the PTA linkers for site-specific release of anticancer drugs into cancer cells.

Electric field stimulation induced neuronal differentiation of filum terminale derived neural progenitor cells.

Adult filum terminale (FT) is an atypical region from where multipotent neural progenitor cells (NPCs) have been isolated. However, poor neuronal differentiation rate of FT-NPCs currently limits their clinical applications. Using custom-designed electric fields (EFs), this study sets up a method to significantly improve neuronal differentiation rate of rat FT-NPCs in vitro. We investigated the influence of EF strength on rat FT-NPCs differentiation. By adding reasonable strength of EF to FT-NPCs, our data shows a significant increase in neuronal differentiation rate. The present innovation provides a novel method of directional differentiation and efficient production of neurons from FT-NPCs in vitro. This improved approach for inducing neuronal differentiation can be applied to future research on autoplastic transplantation.