Correct guidance of the migration of neural progenitor cells (NPCs) is essential for the development and repair of the central nervous system (CNS). Electric field (EF)-guided migration, electrotaxis, has been observed in many cell types. We report here that, in applied EFs of physiological magnitude, embryonic and adult NPCs show marked electrotaxis, which is dependent on the PI3K/Akt pathway. The electrotaxis was also evidenced by ex vivo investigation that transplanted NPCs migrated directionally towards cathode in organotypic spinal cord slice model when treated with EFs. Genetic disruption or pharmacological inhibition of phosphoinositide 3-kinase (PI3K) impaired electrotaxis, whereas EF exposure increased Akt phosphorylation in a growth factor-dependent manner and increased phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels. EF treatments also induced asymmetric redistribution of PIP3, growth factor receptors, and actin cytoskeleton. Electrotaxis in both embryonic and adult NPCs requires epidermal growth factor (EGF) and fibroblast growth factor (FGF). Our results demonstrate the importance of the PI3K/Akt pathway in directed migration of NPCs driven by EFs and growth factors and highlight the potential of EFs to enhance the guidance of various NPC populations in CNS repair therapies.

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 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 condition of TAMM condensation warrants its use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.

Neural stem cells (NSCs) exhibit features that make them suitable candidates for stem cell replacement therapy and spinal cord reconstruction. Magnetic resonance imaging (MRI) offers the potential to track cells in vivo using innovative approaches to cell labeling and image acquisition. In this study, experiments were carried out to optimize the loading condition of magnetic CoPt hollow nanoparticles (CoPt NPs) into neural stem cells and to define appropriate MRI parameters. Both cell viability and multipotency analysis showed that CoPt NPs at a concentration of 16 µg ml(-1) reduced T2 relaxation times in labeled rat NSCs, producing greater contrast on spin echo acquisitions at 4.7 T, yet did not affect cell viability and in vitro differentiation potential compared to controls. After optimizing nanoparticle loading concentrations and labeled cell numbers for MRI detection, CoPt-loaded NSCs were transplanted into organotypic spinal cord slices. The results showed that MRI could efficiently detect low numbers of CoPt-labeled NSCs with the enhanced image contrast. Our study demonstrated that MRI of grafted NSCs labeled with CoPt NPs is a useful tool to evaluate organotypic spinal cord slice models and has potential applications in other biological systems.

Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Cerebrovascular disease such as stroke is one of the most common diseases in the aging population, and neural stem cells (NSCs) transplantation may provide an alternative therapy for cerebral ischemia. However, a hostile microenvironment in the ischemic brain offers is challenging for the survival of the transplanted cells. Considering the neuroprotective role of basic fibroblast growth factor (bFGF), the present study investigated whether bFGF gene-modified NSCs could improve the neurological function deficit after transient middle cerebral artery occlusion (MCAO) in adult male Sprague-Dawley rats. These rats were intravenously injected with modified NSCs (5×10 6 /200 μL) or vehicle 24 h after MCAO. Histological analysis was performed on days 7 and 28 after tMCAO. The survival, migration, proliferation, and differentiation of the transplanted modified C17.2 cells in the brain were improved. In addition, the intravenous infusion of NSCs and bFGF gene-modified C17.2 cells improved the functional recovery as compared to the control. Furthermore, bFGF promoted the C17.2 cell growth, survival, and differentiation into mature neurons within the infarct region. These data suggested that bFGF gene-modified NSCs have the potential to be a therapeutic agent in brain ischemia. www.impactjournals.com/oncotarget/

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 cellpermeable 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.

Amniotic epithelial cells (AECs) were reported to show a neuroprotective effect on neurons, but there was no direct evidence for a functional relationship between neural stem cells (NSCs) and AECs. The aim of this study was to determine whether AECs could stimulate differentiation and expand neurogenesis of NSCs, and whether the roles were due to a diffusible factor or required direct cell-cell contact. AECs were isolated from rat amnion on E14-16 and NSCs were isolated from neocortical tissue. The growth and differentiation of NSCs were compared under different conditions. The results showed that NSCs cultured with FGF-2 proliferated and formed floating neurospheres while those grown in B27 without FGF-2 failed to thrive. Those grown either with AEC conditioned medium or in transwells showed significantly improved survival. Moreover, the neural differentiation and length of neurite were greater in exogenous FGF groups when NSCs were allowed direct contact with AECs. Western blotting, immunocytochemistry and RT-PCR indicated that rat AECs could secrete NT-3 and BDNF. Furthermore, the presence of FGF-2 enhanced the function of AECs. These findings identified that AECs may be regarded as a critical component of NSCs niche and suggested that direct cell-to-cell contact may provide additional and independent support. Such information would circumvent the need for AECs-NSCs co-culture and could potentially facilitate the production of neurons for future clinical applications.

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.

No abstract