A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Self-assembling peptide nanofibrous hydrogel as a promising strategy in nerve repair after traumatic injury in the nervous system

Following injury in central nervous system（CNS）,there are pathological changes in the injured region,which include neuronal death,axonal damage and demyelination,inflammatory response and activation of glial cells.The proliferation of a large number of astrocytes results in the formation of glial scar.

Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells （PSCs） into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group （P〈0.05）. Quantitative real-time PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group （P〈0.01）. ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group＇s PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated （P〈0.01）. The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering.

In situ AFM investigation of dual-mode self-assembling peptide

Nanostructures/patterns formed by biomolecules can produce different physicochemical properties in terms of hydrophobicity, zeta-potential, color, etc., which play paramount roles in life. Peptides, as the main bio-building blocks, can form nanostructures with different functions,either in solutions or on interfaces. Previously, we synthesized a short peptide with the inspiration of an Alzheimer’s disease-related peptide: amyloid β peptide(A-p),namely GAV-9, which can epitaxially self-assemble into regular nanofilaments on liquid-solid interfaces, and it was found that both the hydrophobicity and charge state of the interfaces can significantly influence its assembling behavior. It was also reported that another A-β-containing dipeptide, FF,can self-assemble into nanostructures in solutions. Owing to the close relationship between these two short peptides, it is interesting to conjugate them into a de novo peptide with two separated structural domains and study its self-assembling behavior. To this end, herein we have synthesized the GAV-FF peptide with a sequence of NH2-VGGAVVAGVFF-CONH2 and verified its selfassembling property using the in situ liquid-phase atomic force microscopy. The results show that the GAV-FF peptide can self-assemble into nanofilaments both in solutions and on aqueous-solid interfaces, but with different morphologies. The FF domain accelerates the template-assisted self-assembling(TASA) process of the GAV domain, which in return enhances the solubility of FF in aqueous solutions and further participates in the fibrillization of FF. The current results could help deepen the understanding of the aggregation mechanism of diseaserelated peptides and could also shed light on the strategies to create artificial bio-functional nanostructures/patterns,which hold a significant potential for biomedical applications.

Self-Assembling Peptide as a Candidate Carrier for 5-Fluorouracil

The potential application of a designed self-assembly peptide CH3CO-Pro-Thr-Phe-CysPhe-Lys-Phe-Glu-Pro-NH2（named as P1） as a carrier of 5-Fluorouracil（5-Fu） for controlled release in vitro was studied. 5-Fluorouracil（5-Fu） was selected as a representative anticancer drug due to its extensive use in treating digestive system cancer and breast cancer. The interaction between P1 and 5-Fu was detected by fluorescent quenching experiments and atomic force microscopy（AFM）. The quenching mechanism of 5-Fu and P1 system was dynamic by performing fluorescent quenching experiments at different temperatures. The thermodynamic analysis demonstrated that the interaction between 5-Fu and P1 was hydrophobic interaction. The complexes prepared by the interaction between peptide and 5-Fu appeared as large granular particles of about 20 nm in height under AFM（denoted as5-Fu-P1）, 24 times larger than the original 5-Fu particles. According to the results, an interaction model was proposed. Furthermore, 5-Fu-P1 complexes exhibited an efficient controlled release of 5-Fu in vitro. The research suggested that P1 might be a candidate carrier for drug delivery, providing a substitution agent for 5-Fu.

A neurotrophic peptide-functionalized self-assembling peptide nanofiber hydrogel enhances rat sciatic nerve regeneration

Nerve guidance conduit （NGC） is a potential alternative to autologous nerve for peripheral nerve regeneration. A promising therapeutic strategy is to modify the nerve guidance conduit intraluminal microenvironment using physical and/or chemical guidance cues. In this study, a neurotrophic peptide-functionalized self-assembling peptide nanofiber hydrogel that could promote PC12 cell adhesion, proliferation, and neuronal differentiation in vitro was prefilled in the lumen of a hollow chitosan tube （hCST） to accelerate axonal regeneration in a rat sciatic nerve defect model. The functionalized self-assembling peptide was developed by introducing a neurotrophic peptide （RGI, RGIDKRHWNSQ） derived from brain-derived neurotrophic factor （BDNF） to the C-terminus of the self-assembling peptide RADA16-I （Ac-（RADA）4-CONH2）. Morphological, histological, electrophysiological, and functional analyses demonstrated that the RGI-functionalized, self-assembling, peptide nanofiber hydrogel RAD/RGI could produce a neurotrophic microenvironment that markedly improved axonal regeneration with enhanced re-myelination and motor functional recovery.

Functional self-assembling peptide nanofiber hydrogel for peripheral nerve regeneration

Peripheral nerves are fragile and easily damaged,usually resulting in nervous tissue loss,motor and sensory function loss.Advances in neuroscience and engineering have been significantly contributing to bridge the damage nerve and create permissive environment for axonal regrowth across lesions.We have successfully designed two self-assembling peptides by modifying RADA 16-I with two functional motifs IKVAV and RGD.Nanofiber hydrogel formed when combing the two neutral solutions together,defined as RADA 16-Mix that overcomes the main drawback of RADA16-I associated with low pH.In the present study,we transplanted the RADA 16-Mix hydrogel into the transected rat sciatic nerve gap and effect on axonal regeneration was examined and compared with the traditional RADA16-I hydrogel.The regenerated nerves were found to grow along the walls of the large cavities formed in the graft of RADA16-I hydrogel,while the nerves grew into the RADA 16-Mix hydrogel toward distal position.RADA 16-Mix hydrogel induced more axons regeneration and Schwann cells immigration than RADA16-I hydrogel,resulting in better functional recovery as determined by the gait-stance duration percentage and the formation of new neuromuscular junction structures.Therefore,our results indicated that the functional SAP RADA16-Mix nanofibrous hydrogel provided a better environment for peripheral nerve regeneration than RADA16-I hydrogel and could be potentially used in peripheral nerve injury repair.

Aligned fibrin/functionalized self-assembling peptide interpenetrating nanofiber hydrogel presenting multi-cues promotes peripheral nerve functional recovery

Nerve guidance conduits with hollow lumen fail to regenerate critical-sized peripheral nerve defects(15 mm in rats and 25 mm in humans),which can be improved by a beneficial intraluminal microenvironment.However,individual cues provided by intraluminal filling materials are inadequate to eliminate the functional gap between regenerated nerves and normal nerves.Herein,an aligned fibrin/functionalized self-assembling peptide(AFG/fSAP)interpenetrating nanofiber hydrogel that exerting synergistic topographical and biochemical cues for peripheral nerve regeneration is constructed via electrospinning and molecular self-assembly.The hydrogel possesses an aligned structure,high water content,appropriate mechanical properties and suitable biodegradation capabilities for nerve repair,which enhances the alignment and neurotrophin secretion of primary Schwann cells(SCs)in vitro,and successfully bridges a 15-mm sciatic nerve gap in rats in vivo.The rats transplanted with the AFG/fSAP hydrogel exhibit satisfactory morphological and functional recovery in myelinated nerve fibers and innervated muscles.The motor function recovery facilitated by the AFG/fSAP hydrogel is comparable with that of autografts.Moreover,the AFG/fSAP hydrogel upregulates the regeneration-associated gene expression and activates the PI3K/Akt and MAPK signaling pathways in the regenerated nerve.Altogether,the AFG/fSAP hydrogel represents a promising approach for peripheral nerve repair through an integration of structural guidance and biochemical stimulation.

Self-assembling peptide nanofiber hydrogels for central nervous system regeneration

Central nervous system （CNS） presents a complex regeneration problem due to the inability of central neurons to regenerate correct axonal and dendritic connections. However, recent advances in developmental neurobiology, cell signaling, cell-matrix interaction, and biomaterials technologies have forced a reconsideration of CNS regeneration potentials from the viewpoint of tissue engineering and regenerative medicine. The applications of a novel tissue regeneration-inducing biomaterial and stem cells are thought to be critical for the mission. The use of peptide nanoflber hydrogels in cell therapy and tissue engineering offers promising perspectives for CNS regeneration. Self-assembling peptide undergo a rapid transformation from liquid to gel upon addition of counterions or pH adjustment, directly integrating with the host tissue. The peptide nanofiber hydrogels have mechanical properties that closely match the native central nervous extracellular matrix, which could enhance axonal growth. Such materials can provide an optimal three dimensional microenvironment for encapsulated cells. These materials can also be tailored with bioactive motifs to modulate the wound environment and enhance regeneration. This review intends to detail the recent status of selfassembling peptide nanoflber hydrogels for CNS regeneration.

Novel umami peptides from two Termitomyces mushrooms and molecular docking to the taste receptor T1R1/T1R3

Wild edible Termitomyces mushrooms are popular in Southwest China and umami is important flavor qualities of edible mushrooms.This study aimed to understand the umami taste of Termitomyces intermedius and Termitomyces aff.bulborhizus.Ten umami peptides from aqueous extracts were separated using a Sephadex G-15 gel filtration chromatography.The intense umami fraction was evaluated by both sensory evaluation and electronic tongue.They were identified as KLNDAQAPK,DSTDEKFLR,VGKGAHLSGEH,MLKKKKLA,SLGFGGPPGY,TVATFSSSTKPDD,AMDDDEADLLLLAM,VEDEDEKPKEK,SPEEKKEEET and PEGADKPNK.Seven peptides,except VEDEDEKPKEK,SPEEKKEEET and PEGADKPNK were selectively synthesized to verify their taste characteristics.All these 10 peptides had umami or salt taste.The 10 peptides were conducted by molecular docking to study their interaction with identified peptides and the umami taste receptor T1R1/T1R3.All these 10 peptides perfectly docked the active residues in the T1R3 subunit.Our results provide theoretical basis for the umami taste and address the umami mechanism of two wild edible Termitomyces mushrooms.

Three novel umami peptides derived from the alcohol extract of the Pacific oyster(Crassostrea gigas):identification,characterizations and interactions with T1R1/T1R3 taste receptors

Oyster(Crassostrea gigas),the main ingredient of oyster sauce,has a strong umami taste.In this study,three potential umami peptides,FLNQDEEAR(FR-9),FNKEE(FE-5),and EEFLK(EK-5),were identified and screened from the alcoholic extracts of the oyster using nano-HPLC-MS/MS analysis,i Umami-Scoring Card Method(i Umami-SCM)database and molecular docking(MD).Sensory evaluation and electronic tongue analysis were further used to confirm their tastes.The threshold of the three peptides ranged from 0.38 to 0.55 mg/m L.MD with umami receptors T1R1/T1R3 indicated that the electrostatic interaction and hydrogen bond interaction were the main forces involved.Besides,the Phe592 and Gln853 of T1R3 were the primary docking site for MD and played an important role in umami intensity.Peptides with two Glu residues at the terminus had stronger umami,especially at the C-terminus.These results contribute to the understanding of umami peptides in oysters and the interaction mechanism between umami peptides and umami receptors.

Cross-linked self-assembling peptide scaffolds

Self-assembling peptides （SAPs） are synthetic bioinspired biomaterials that can be feasibly multi-functionalized for cell transplantation and/or drug delivery therapies. Despite their superior biocompatibility and ease of scaling-up for production, they are unfortunately hampered by weak mechanical properties due to transient non-covalent interactions among and within the self-assembled peptide chains, thus limiting their potential applications as fillers, hemostat solutions, and fragile scaffolds for soft tissues. Here, we have developed and characterized a cross-linking strategy that increases both the stiffness and the tailorability of SAP hydrogels, enabling the preparation of transparent flexible threads, discs, channels, and hemispherical constructs. Empirical and computational results, in close agreement with each other, confirmed that the cross-linking reaction does not affect the previously self-assembled secondary structures. In vitro tests also provided a first hint of satisfactory biocompatibility by favoring viability and differentiation of human neural stem cells. This work could bring self-assembling peptide technology to many applications that have been precluded so far, especially in regenerative medicine.

Neutrophil peptide 1 accelerates the clearance of degenerative axons during Wallerian degeneration by activating macrophages after peripheral nerve crush injury

Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Nitric oxide-generating self-assembling peptide hydrogel coating for enhancing hemocompatibility of blood-contacting devices

Thrombosis is the major stumbling block to the clinical application of blood-contacting devices.Herein,a quick and easy surface engineering strategy of hydrogel coating with the therapeutic gas nitric oxide(NO)generation was reported to realize up-regulation of cyclic guanosine monophosphate(c GMP)and improve hemocompatibility for diverse metal materials.We first introduce the active centre selenocysteine of glutathione peroxidase(GPx)to the self-assembling peptide(RADA)4,obtaining a functionalized hydrogel.Then the hydrogel is directly coated on the 316L stainless steel(SS)for catalytically generating NO from endogenous s-nitrosothiols(RSNO).The generated NO endows the coated surface with regulation of platelet behavior and reduction of plasmatic coagulation activation and complement system activation,hence improving antithrombotic ability in vitro and ex vivo.Overall,our NO-generating hydrogel coating surface engineering strategy provides a novel solution to remove the obstacle about thrombosis of blood-contacting devices in clinic.

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations.In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide （SAP） RADA （AcN-RADARADARADARADA-CONH2） nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.

MiR-107-3p/Atp6v0e1 contributes to protective effects of two selenium-containing peptides,TSeMMM and SeMDPGQQ on lead-induced neurotoxicity

Two selenium(Se)-containing peptides from Se-enriched rice,TSeMMM and SeMDPGQQ,possess neuroprotective potency against lead(Pb2+)-induced cytotoxicity.However,the crosstalk between mRNA and microRNAs(miRNA)involved in the neuroprotection mechanism remains to be elucidated.In this study,RNA-sequencing and miRNA-sequencing were used to independently identify differentially expressed mRNAs and small RNAs profiles in Pb^(2+)-treated primary fetal rat cortical neurons and then the correlated miRNA-mRNA target pairs were obtained.It was found that 34 mRNAs related to oxidative phosphorylation could be reversed by pretreatment of TSeMMM and SeMDPGQQ.The protective effect of TSeMMM and SeMDPGQQ was mediated by upregulation of miR-107-3p,which downregulates the ATPase H+transporting V0 subunit e1(Atp6v0e1)mRNA level.A zebrafish model was applied to verify the relevance between the targeted mRNA and miRNA by real-time quantitative PCR.The results indicated that miR-107-3p was a potential therapeutic target to achieve neuroprotection of Se-containing peptides via stimulation of Atp6v0e1.

Food-derived protein hydrolysates and peptides:anxiolytic and antidepressant activities,characteristics,and mechanisms

Globally,the prevalence of anxiety and depression has reached epidemic proportions.Food-derived protein hydrolysates and peptides delivered through dietary supplementation can avoid the negative risks associated with traditional pharmaceuticals while delivering superior anxiolytic and antidepressant effects.This review summarizes current research on food-derived anxiolytic and antidepressant protein hydrolysates and peptides,and subsequently analyses their physicochemical characteristics and elaborates on their mechanisms.The aim of this work is to contribute to the in-depth study and provide a theoretical foundation for the development of related products to better serve patients with anxiety and depression.

Food-derived bio-functional peptides for the management of hyperuricemia and associated mechanism

Hyperuricemia,a metabolic disorder related to uric acid metabolism dysregulation,has become a common metabolic disease worldwide,due to changes in lifestyle and dietary structure.In recent years,owing to their high activity and few adverse effects,food-derived active peptides used as functional foods against hyperuricemia have attracted increasing attention.This article aims to focus on the challenge associated with peptide-specific preparation methods development,functional components identification,action mechanism(s)clarification,and bioavailability improvement.The current review proposed recent advances in producing the food-derived peptides with high anti-hyperuricemia activity by protein source screening and matched enzymatic hydrolysis condition adjusting,increased the knowledge about strategies to search antihyperuricemia peptides with definite structure,and emphasized the necessity of combining computer-aided approaches and activity evaluations.In addition,novel action mechanism mediated by gut microbiota was discussed,providing different insights from classical mechanism.Moreover,considering that little attention was paid previously on the structure-activity relationships of anti-hyperuricemia peptides,we collected the sequences from published studies and make a preliminary summary about the structure-activity relationships,which in turn provided guides for enzymatic hydrolysis optimization and bioavailability improvement.Hopefully,this article could promote the development,application and commercialization of food-derived anti-hyperuricemia peptides in the future.

Glucagon-like peptide 1 receptor activation:anti-inflammatory effects in the brain

The glucagon-like peptide 1 is a pleiotropic hormone that has potent insulinotropic effects and is key in treating metabolic diseases such as diabetes and obesity.Glucagon-like peptide 1 exerts its effects by activating a membrane receptor identified in many tissues,including diffe rent brain regions.Glucagon-like peptide 1 activates several signaling pathways related to neuroprotection,like the support of cell growth/survival,enhancement promotion of synapse formation,autophagy,and inhibition of the secretion of proinflammatory cytokines,microglial activation,and apoptosis during neural morphogenesis.The glial cells,including astrocytes and microglia,maintain metabolic homeostasis and defe nse against pathogens in the central nervous system.After brain insult,microglia are the first cells to respond,followed by reactive astrocytosis.These activated cells produce proinflammato ry mediators like cytokines or chemokines to react to the insult.Furthermore,under these circumstances,mic roglia can become chro nically inflammatory by losing their homeostatic molecular signature and,consequently,their functions during many diseases.Several processes promote the development of neurological disorders and influence their pathological evolution:like the formation of protein aggregates,the accumulation of abnormally modified cellular constituents,the formation and release by injured neurons or synapses of molecules that can dampen neural function,and,of critical impo rtance,the dysregulation of inflammato ry control mechanisms.The glucagonlike peptide 1 receptor agonist emerges as a critical tool in treating brain-related inflammatory pathologies,restoring brain cell homeostasis under inflammatory conditions,modulating mic roglia activity,and decreasing the inflammato ry response.This review summarizes recent advances linked to the anti-inflammato ry prope rties of glucagon-like peptide 1 receptor activation in the brain related to multiple sclerosis,Alzheimer’s disease,Parkinson’s disease,vascular dementia,or chronic migraine.