Multifunctional Peptide Research Articles

Structural Plasticity of LL-37 Indicates Elaborate Functional Adaptation Mechanisms to Bacterial Target Structures.

The human cathelicidin LL-37 is a multifunctional peptide of the human innate immune system. Among the various functions of LL-37, its antimicrobial activity is important in controlling the microorganisms of the human body. The target molecules of LL-37 in bacteria include membrane lipids, lipopolysaccharides (LPS), lipoteichoic acid (LTA), proteins, DNA and RNA. In this mini-review, we summarize the entity of LL-37 structural data determined over the last 15 years and specifically discuss features implicated in the interactions with lipid-like molecules. For this purpose, we discuss partial and full-length structures of LL-37 determined in the presence of membrane-mimicking detergents. This constantly growing structural database is now composed of monomers, dimers, tetramers, and fiber-like structures. The diversity of these structures underlines an unexpected plasticity and highlights the conformational and oligomeric adaptability of LL-37 necessary to target different molecular scaffolds. The recent co-crystal structures of LL-37 in complex with detergents are particularly useful to understand how these molecules mimic lipids and LPS to induce oligomerization and fibrillation. Defined detergent binding sites provide deep insights into a new class of peptide scaffolds, widening our view on the fascinating world of the LL-37 structural factotum. Together, the new structures in their evolutionary context allow for the assignment of functionally conserved residues in oligomerization and target interactions. Conserved phenylalanine and arginine residues primarily mediate those interactions with lipids and LPS. The interactions with macromolecules such as proteins or DNA remain largely unexplored and open a field for future studies aimed at structures of LL-37 complexes. These complexes will then allow for the structure-based rational design of LL-37-derived peptides with improved antibiotic properties.

Interaction of LL-37 human cathelicidin peptide with a model microbial-like lipid membrane

The only representative of cathelicidin peptides in humans is LL-37, a multifunctional antimicrobial peptide (AMP) that is a part of the innate immune response. Details of the LL-37 direct activity against pathogens are not well understood at the molecular level. Here, we present research on the mechanism of interaction between LL-37 and a model multicomponent bilayer lipid membrane (BLM), mimicking microbial cell membrane. Electrochemical impedance spectroscopy (EIS), high-resolution atomic force microscopy (AFM) imaging, and polarization-modulation infrared reflection–absorption spectroscopy (PM-IRRAS) were applied to study the peptide influence on a model microbial-like membrane. We show that LL-37 causes changes in the phospholipid molecules conformation and orientation, leading to membrane disintegration, significantly affecting the membrane electrical parameters, such as capacitance and resistance. High-resolution AFM imaging shows topographical and mechanical effects of such disintegration, while PM-IRRAS data indicates that introduction of LL-37 causes changes in the phospholipid acyl chains from all-trans to gauche conformations. Moreover, the presence of LL-37 significantly alters the value of the phospholipid tilt angle. Altogether, our results suggest a “carpet” membrane dissolution followed by a detergent-like membrane disruption mechanism upon LL-37 activity. This research gives a novel insight into the understanding of LL-37 influence on multicomponent model membranes and a promising contribution to the development of LL-37-derived therapeutic agents against drug-resistant bacteria.

Endothelin receptor antagonists for the treatment of diabetic and nondiabetic chronic kidney disease.

To summarize new clinical findings of endothelin receptor antagonists (ERA) in various etiologies of kidney disease targeted in clinical trials. Endothelin-1 is a multifunctional peptide with potential relevance to glomerular and tubulointerstitial kidney diseases. The phase 3 SONAR trial demonstrated a significant reduction in clinically relevant kidney outcomes for patients with diabetic kidney disease (DKD) after long-term treatment with the ERA, atrasentan, in addition to blockade of the renin-angiotensin-aldosterone system. Promising preclinical disease models and small clinical trials in non-DKD resulted in the initiation of phase 3 trials investigating the effects of long-term treatment with ERA in patients with immunoglobulin A (IgA) nephropathy and focal segmental glomeruloscelerosis (FSGS). The mechanisms by which ERA protects the kidneys have been extensively studied with evidence for the protection of tubule cells, podocytes, mesangial cells, the endothelial glycocalyx, and a reduction in glomerular perfusion pressure. The occurrence of fluid retention during ERA treatment, particularly in susceptible populations, necessitates strategies to support safe and effective treatment. Treatment with ERA induces long-term kidney protection in DKD. Phase 3 trials are underway to investigate ERA effects in patients with IgA nephropathy and FSGS.

Adrenomedullin Ameliorates Pulmonary Fibrosis by Regulating TGF-ß-Smads Signaling and Myofibroblast Differentiation.

Pulmonary fibrosis is an irreversible, potentially fatal disease. Adrenomedullin (AM) is a multifunctional peptide whose activity is regulated by receptor activity-modifying protein 2 (RAMP2). In the present study, we used the bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the pathophysiological significance of the AM-RAMP2 system in the lung. In heterozygous AM knockout mice (AM+/-), hydroxyproline content and Ashcroft scores reflecting the fibrosis severity were significantly higher than in wild-type mice (WT). During the acute phase after BLM administration, FACS analysis showed significant increases in eosinophil, monocyte, and neutrophil infiltration into the lungs of AM+/-. During the chronic phase, fibrosis-related molecules were upregulated in AM+/-. Notably, nearly identical changes were observed in RAMP2+/-. AM administration reduced fibrosis severity. In the lungs of BLM-administered AM+/-, the activation level of Smad3, a receptor-activated Smad, was higher than in WT. In addition, Smad7, an antagonistic Smad, was downregulated and microRNA-21, which targets Smad7, was upregulated compared to WT. Isolated AM+/- lung fibroblasts showed less proliferation and migration capacity than WT fibroblasts. Stimulation with TGF-β increased the numbers of α-SMA-positive myofibroblasts, which were more prominent among AM+/- cells. TGF-β-stimulated AM+/- myofibroblasts were larger and exhibited greater contractility and extracellular matrix production than WT cells. These cells were α-SMA (+), F-actin (+), and Ki-67(-) and appeared to be nonproliferating myofibroblasts (non-p-MyoFbs), which contribute to the severity of fibrosis. Our findings suggest that in addition to suppressing inflammation, the AM-RAMP2 system ameliorates pulmonary fibrosis by suppressing TGF-β-Smad3 signaling, microRNA-21 activity and differentiation into non-p-MyoFbs.

Barbel steed (Hemibarbus labeo) NK-lysin protects against Aeromonas hydrophila infection via immunomodulatory activity

NK-lysins (NKLs) are a family of multifunctional antimicrobial peptides that have activity against various microorganisms. However, the immunomodulatory activity of NKL in fish remains unclear. In this study, the cDNA sequence of barbel steed (Hemibarbus labeo) NKL gene was cloned. Barbel steed NKL amino acid sequence comprised a signal peptide and a mature peptide. The saposin B domain in the mature peptide has six conserved cysteines that form three disulfide bonds. Phylogenetic analysis showed that the barbel steed NKL was most closely related to that of the common carp (Cyprinus carpio) NKL. Differential expression analysis showed that the barbel steed NKL gene was expressed in all tested tissues, with the highest expression in the spleen. In response to Aeromonas hydrophila infection, NKL was significantly upregulated in the liver, spleen, head kidney, and gill. The barbel steed NKL showed strong antibacterial activity against Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, and Listeria monocytogenes. However, NKL had no antibacterial activity against the pathogenic bacteria A. hydrophila. Lactate dehydrogenase release assays showed that NKL damaged the V. parahaemolyticus cell membrane. NKL significantly increased barbel steed survival rate after A. hydrophila infection and upregulated IL-1β and TNF-α expression in the spleen and head kidney. NKL induced monocyte/macrophage chemotaxis and enhanced the respiratory burst and proinflammatory cytokine expression. Our study shows that fish NKL exhibits immunomodulatory effects and protects the host from pathogenic infections independent of direct bacterial clearance.

β-Defensins from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus): Molecular characterization and phylogenetic analysis.

Antimicrobial peptides (AMPs) are biologically active molecules involved in host defense present in a variety of organisms. They arean integral component of innate immunity, forming a front line of defense against potential pathogens, including antibiotic-resistant ones. Fishes are proven to be a prospective source of AMPs as they are constantly being challenged by a variety of pathogens and the AMPs are reported to play an inevitable role in fish immunity. Among them, β-defensins form one of the most studied multifunctional peptides with early evolutionary history and recently being considered as host defense peptides. The present study highlights the first-ever report on β-defensin AMP sequences from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus). A 192bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) comprising a 20 aa signal peptide region at the N-terminal was obtained from the mRNA of gill tissue of both P. microps and P. georgianus by RT-PCR. These peptide sequences when characterized in silico at the molecular level revealed a 43 aa cationic mature peptide with the signature intra-molecular disulphide bonded cysteine residue pattern ascertaining its β-defensin identity, further confirmed by phylogenetic analysis. The data collected will pave the way for further research on varied facets of the peptide-like, tissue level expressions, antimicrobial activities on commonly encountered pathogens, and its feasibility as atherapeutant in the aquaculture scenario.

Ratiometric Antifouling Electrochemical Biosensors Based on Multifunctional Peptides and MXene Loaded with Au Nanoparticles and Methylene Blue.

A universal strategy for the construction of ratiometric antifouling electrochemical biosensors was developed based on multifunctional peptides and 2D nanomaterial MXene loaded with gold nanoparticles (AuNPs) and methylene blue (MB). The nanocomposite of MXene loaded with AuNPs and MB (MXene-Au-MB) exhibited excellent conductivity, where the AuNPs were able to capture biomolecules containing sulfhydryl terminus, and the MB molecules were used to generate electrochemical signal. The MXene-Au-MB was fixed on the electrode surface by Nafion, and the anchored peptide captured the electrochemical signal probe carboxyl-modified ferrocene (Fc) to construct an electrochemical biosensor. The multifunctional peptide containing the anchoring, antifouling, and recognizing sequences endowed the sensing surface not only the assaying function but also the capability to resist nonspecific adsorption from complex samples. In the biosensing system, with the increase in the target concentration, the electrochemical signal of MB remained constant, whereas the electrochemical signal of Fc gradually decreased, and the ratiometric detection strategy greatly improved the accuracy of the biosensor. In the presence of a model target prostate-specific antigen (PSA), the recognizing sequence was recognized and cleaved, and the ratiometric signal of Fc and MB indicated the concentration of PSA accurately and sensitively, with a detection range from 5 pg/mL to 10 ng/mL and a limit of detection of 0.83 pg/mL. Electrochemical biosensors based on the MXene-Au-MB and multifunctional peptides possessed high selectivity, accuracy, and sensitivity even in real complex biological samples because of the excellent antifouling ability of the peptide. More importantly, the assaying of other targets can be easily realized with a similar biosensing strategy by changing the recognition sequence of the multifunctional peptide, and the detection of thrombin (TB) has also been achieved in this work.

Improvement of Vascular Function by Knockdown of Salusin-β in Hypertensive Rats via Nitric Oxide and Reactive Oxygen Species Signaling Pathway.

PurposeSalusin-β, a multifunctional vasoactive peptide, has a potentially important function in the pathological development of hypertension. However, the exact functional role of salusin-β and the underlying mechanism in this process are still not fully understood. The current study aimed to investigate the effects of silencing salusin-β on vascular function and vascular remodeling, as well as its signaling pathways in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).MethodsSilencing salusin-β was performed by caudal vein injection of adenovirus expressing salusin-β short hairpin RNA (shRNA). Acetylcholine (ACh)-induced endothelium-dependent relaxation was used to evaluate vasodilator function, and high K+ solution-induced constriction was used to evaluate vasoconstriction function.ResultsSalusin-β levels in plasma and its protein expression in mesenteric artery (MA), coronary artery (CA), and pulmonary artery (PA) of SHR were higher than those in WKY. The salusin-β level and expression were decreased effectively by salusin-β shRNA. Knockdown of salusin-β decreased arterial blood pressure (ABP) and high K+ solution-induced vascular constrictions, and improved the endothelium-dependent relaxation and vascular remodeling in SHR. The improved effect of silencing salusin-β on ACh-induced relaxation in SHR was almost blocked by the nitric oxide synthase (NOS) inhibitor L-NAME. Compared to WKY, the endothelial NOS (eNOS) activity and level, and nitric oxide (NO) level were decreased, while NAD(P)H oxidase activity and reactive oxygen species (ROS) levels in MA, CA, and PA of SHR were increased, which were all redressed by salusin-β knockdown.ConclusionThese results indicate that knockdown of salusin-β improves endothelium-dependent vascular relaxation and vascular remodeling and decreases ABP and vasoconstriction in SHR, which might be accomplished by increasing eNOS activation and NO release while inhibiting NAD(P)H oxidase derived-ROS generation. Scavenging salusin-β improves vascular function and then prevents the development and progression of vasculopathy of hypertension.

β-Thymosin is an essential regulator of stem cell proliferation and neuron regeneration in planarian (Dugesia japonica)

β-Thymosin is a multifunctional peptide ubiquitously expressed in vertebrates and invertebrates. Many studies have found β-thymosin is critical for wound healing, angiogenesis, cardiac repair, hair regrowth, and anti-fibrosis in vertebrates, and plays an important role in antimicrobial immunity in invertebrates. However, whether β-thymosin participates in the regeneration of organisms is still poorly understood. In this study, we identified a β-thymosin gene in Dugesia japonica which played an important role in stem cell proliferation and neuron regeneration during the tissue repair process in D. japonica. Sequencing analysis showed that β-thymosin contained two conserved β-thymosin domains and two actin-binding motifs, and had a high similarity with other β-thymosins of invertebrates. In situ or fluorescence in situ hybridization analysis revealed that Djβ-thymosin was co-localized with DjPiWi in the neoblast cells of intact adult planarians and the blastema of regenerating planarians, suggesting Djβ-thymosin has a potential function of regeneration. Disruption Djβ-thymosin by RNA interference results in a slightly curled up head of planarian and stem cell proliferation defects. Additionally, we found that, upon amputation, Djβ-thymosin RNAi-treated animals had impaired regeneration ability, including impaired blastema formation, delayed eyespot formation, decreased brain area, and disrupted central CNS formation, implying Djβ-thymosin is an essential regulator of stem cell proliferation and neuron regeneration.

Multifunctional peptide-conjugated nanocarriers for pulp regeneration in a full-length human tooth root

Dental pulp is a highly vascularized tissue, situated in an inextensible environment surrounded by rigid dentinal walls. The pulp receives its blood supply solely from the small apical foramen of a tooth root. Due to the unique anatomy that controls nutrition supply, regeneration of pulp tissue in a full-length tooth root has long been a challenge in regenerative endodontics. In this study, we designed and synthesized a multifunctional peptide-conjugated, pH-sensitive, non-viral gene vector for fast revascularization and pulp regeneration in a full-length human tooth root. The multifunctional peptide was designed to have distinctive features, including a cell-penetrating peptide to enhance cellular uptake, a nuclear localization signal peptide to assist in the translocation of an angiogenic gene into the nucleus, and a fluorescent tryptophan residue to visualize and quantify the transfection efficiency. Furthermore, a pH-sensitive dimethylmaleic anhydride (DMA) was integrated with the multifunctional peptide to enhance the transfected gene complex to escape from endosomes/lysosomes after internalization. In vitro experiments showed that the multifunctional non-viral gene vector significantly increased internalization and gene transfection efficiency as well as reduced cytotoxicity. After dental pulp stem cells (DPSCs) were transfected with the multifunctional gene vector/pVEGF complexes, the expression of VEGF from the DPSCs was upregulated for more than eight folds, which in turn greatly enhanced endothelial cell migration and vascular-like tube formation. Six weeks after implantation, the VEGF-transfected DPSCs accelerated new blood vessel formation and the regenerated pulp tissue occupied most of the area in the canal of a full-length human tooth root. The multifunctional peptide conjugated non-viral gene delivery is a safe and effective approach for regenerative endodontics. Statement of SignificancePulp regeneration in a full-length tooth root canal has long been a challenge in regenerative endodontics. This is due to the unique root anatomy that allows the blood supply of the tooth root only from a small apical foramen (< 1 mm), leading to a severe barrier for revascularization during pulp regeneration. In this work, we designed a multifunctional peptide-conjugated, pH-sensitive, non-viral gene vector to address this challenge. Our work shows that the peptide-conjugated system was an excellent carrier for fast revascularization and pulp tissue regeneration in a full-length toot root. This study will interest the multidisciplinary readership in gene delivery, biomaterials, and dental/craniofacial tissue engineering community.

Structure\u2013function engineering of novel fish gelatin-derived multifunctional peptides using high-resolution peptidomics and bioinformatics

The multifunctional properties of fish gelatin hydrolysates have not been completely elucidated. Here, the biological characterization of these peptides was performed to engineer multifunctional peptides. Bioactive peptides were produced from mackerel byproducts via successive enzymatic hydrolysis reactions using subtilisin A and actinidin as microbial and herbal proteases. The antibacterial activity against both gram-negative and -positive food-borne pathogens, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae, as well as the inhibitory potential of angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), was accessed in vitro. The synthesized peptides demonstrated multifunctional properties, which were further confirmed by in silico protocols. The ACE and DPP-IV inhibitory (IC50) values of P1, P2, and P3 were 0.92 and 0.87, 0.51 and 0.93, 0.78 and 1.16 mg mL−1, respectively. Moreover, the binding energy was sufficient for all three peptides to inhibit both ACE and DPP-IV enzymes with excellent three-dimensional conformation (RMSD = 0.000) for all six docking mechanisms.

A Multifunctional Peptide From Bacillus Fermented Soybean for Effective Inhibition of SARS-CoV-2 S1 Receptor Binding Domain and Modulation of Toll Like Receptor 4: A Molecular Docking Study.

Fermented soybean products are traditionally consumed and popular in many Asian countries and the northeastern part of India. To search for potential agents for the interruption of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike glycoprotein 1 (S1) and human angiotensin-converting enzyme 2 (ACE2) receptor interactions, the in silico antiviral prospective of peptides identified from the proteome of kinema was investigated. Soybean was fermented using Bacillus licheniformis KN1G, Bacillus amyloliquefaciens KN2G and two different strains of Bacillus subtilis (KN2B and KN2M). The peptides were screened in silico for possible antiviral activity using two different web servers (AVPpred and meta-iAVP), and binding interactions of selected 44 peptides were further explored against the receptor-binding domain (RBD) of the S1 protein (PDB ID: 6M0J) by molecular docking using ZDOCK. The results showed that a peptide ALPEEVIQHTFNLKSQ (P13) belonging to B. licheniformis KN1G fermented kinema was able to make contacts with the binding motif of RBD by blocking specific residues designated as critical (GLN493, ASN501) in the binding of human angiotensin-converting enzyme 2 (ACE2) cell receptor. The selected peptide was also observed to have a significant affinity towards human toll like receptor 4 (TLR4)/Myeloid Differentiation factor 2 (MD2) (PDB ID: 3FXI) complex known for its essential role in cytokine storm. The energy properties of the docked complexes were analyzed through the Generalized Born model and Solvent Accessibility method (MM/GBSA) using HawkDock server. The results showed peptidyl amino acids GLU5, GLN8, PHE11, and LEU13 contributed most to P13-RBD binding. Similarly, ARG90, PHE121, LEU61, PHE126, and ILE94 were appeared to be significant in P13-TLR4/MD2 complex. The findings of the study suggest that the peptides from fermented soy prepared using B. licheniformis KN1G have better potential to be used as antiviral agents. The specific peptide ALPEEVIQHTFNLKSQ could be synthesized and used in combination with experimental studies to validate its effect on SARS-CoV-2-hACE2 interaction and modulation of TLR4 activity. Subsequently, the protein hydrolysate comprising these peptides could be used as prophylaxis against viral diseases, including COVID-19.

Multifunctional Tyrosinase Inhibitor Peptides with Copper Chelating, UV-Absorption and Antioxidant Activities: Kinetic and Docking Studies.

Nature-derived tyrosinase inhibitors are of great industrial interest. Three monophenolase inhibitor peptides (MIPs) and three diphenolase inhibitor peptides (DIPs) from a previous study were investigated for their in vitro tyrosinase inhibitory effects, mode of inhibition, copper-chelating activity, sun protection factor (SPF) and antioxidant activities. DIP1 was found to be the most potent tyrosinase inhibitor (IC50 = 3.04 ± 0.39 mM), which could be due to the binding interactions between its aromatic amino acid residues (Y2 and D7) with tyrosinase hotspots (H85, V248, H258, H263, F264, R268, V283 and E322) and its ability to chelate copper ion within the substrate-binding pocket. The conjugated planar rings of tyrosine and tryptophan may interact with histidine within the active site to provide stability upon enzyme-peptide binding. This postulation was later confirmed as the Lineweaver–Burk analysis had identified DIP1 as a competitive inhibitor and DIP1 also showed 36.27 ± 1.17% of copper chelating activity. In addition, DIP1 provided the highest SPF value (11.9 ± 0.04) as well as ferric reducing antioxidant power (FRAP) (5.09 ± 0.13 mM FeSO4), 2,2′–azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (11.34 ± 0.90%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (29.14 ± 1.36%) free radical scavenging activities compared to other peptides. These results demonstrated that DIP1 could be a multifunctional anti-tyrosinase agent with pharmaceutical and cosmeceutical applications.

Isolation and characterization of oligopeptides with vascular disease suppression effects derived from wheat gluten.

Wheat gluten was hydrolyzed with both alkaline protease and neutral protease to produce high-protein and low-wheat-weight oligopeptides (WOP), which was subjected to a multistage purification. Then, high performance liquid chromatography was applied to separate WOP. In order to identify WOP sequences, six major fractions were gathered for mass spectrometry. A total of 15 peptides were synthesized for further in vitro analyses of their antithrombotic activity, vasorelaxation activity, and cholesterol reducing activity. Two antithrombotic peptides (ILPR and ILR), three vasorelaxant peptides (VN, FPQ, and FR), and four cholesterol-lowering peptides (QRQ, ILPR, FPQ, and ILR) were identified. These active peptides in WOP were also quantified. These peptides are novel candidate peptides with vascular disease suppressing effects. The results indicate WOP as good protein sources for multifunctional peptides.

Evaluating the Multifunctionality of a New Modulator of Zinc-Induced Aβ Aggregation Using a Novel Computational Approach.

The high concentration of zinc metal ions in Aβ aggregations is one of the most cited hallmarks of Alzheimer's disease (AD), and several substantial pieces of evidence emphasize the key role of zinc metal ions in the pathogenesis of AD. In this study, while designing a multifunctional peptide for simultaneous targeting Aβ aggregation and chelating the zinc metal ion, a novel and comprehensive approach is introduced for evaluating the multifunctionality of a multifunctional drugs based on computational methods. The multifunctional peptide consists of inhibitor and chelator domains, which are included in the C-terminal hydrophobic region of Aβ, and the first four amino acids of human albumin. The ability of the multifunctional peptide in zinc ion chelation has been investigated using molecular dynamics (MD) simulations of the peptide-zinc interaction for 300 ns, and Bennett's acceptance ratio (BAR) method has been used to accurately calculate the chelation free energy. Data analysis demonstrates that the peptide chelating domain can be stably linked to the zinc ion. Besides, the introduced method used for evaluating chelation and calculating the free energy of peptide binding to zinc ions was successfully validated by comparison with previous experimental and theoretical published data. The results indicate that the multifunctional peptide, coordinating with the zinc metal ion, can be effective in Aβ inhibition by preserving the native helical structure of the Aβ42 monomer as well as disrupting the β-sheet structure of Aβ42 aggregates. Detailed assessments of the Aβ42-peptide interactions elucidate that the inhibition of Aβ is achieved by considerable hydrophobic interactions and hydrogen bonding between the multifunctional peptide and the hydrophobic Aβ regions, along with interfering in stable bridges formed inside the Aβ aggregate.

Multifunctional peptides based on low molecular weight protamine (LMWP) in the structure of polyplexes and lipopolyplexes: Design, preparation and gene delivery characterization

The development of an effective and safe gene carrier is one of the main challenges in gene therapy. In this study, we designed two new cationic peptides (VV45 and VV32) based on low molecular weight protamine (LMWP) which were upgraded by adding a histidine tag (H10) and nuclear localization signal (NLS) peptide and evaluated their transfection efficiency and cytotoxicity in the formulation of Peptide-DNA (PD) and Liposome-Peptide-DNA (LPD) complexes. Using the gel retardation assay for assessment of condensation ability, three carriers to plasmid (C/P) ratios of 1, 3, and 6 for PD and LPD formulations were selected, and prepared vectors were evaluated in terms of size distribution and zeta potential. All vectors in deionized water (DW) had a size below 200 nm (PD complexes: 65–120 nm, and LPD complexes: 140–185 nm) and a positive zeta potential (PD: 3–15 mV, and LPD: 32–48 mV). Generally, the LPD complexes revealed higher GFP expression than PD complexes, up to four-folds. Compared to,polyethyleneimine 25 kDa, as a transfection standard, the LPD VV45 at C/P ratios of 3, 6 and LPD VV32 at C/P ratio of 6 had a higher transfection. In terms of cytotoxicity, PD complexes have no cytotoxicity, and at the LPD complexes, increasing in C/P ratio decreased the cell viability to 30–50%. So newly designed peptides are effective gene carriers in the formulation of LPD with a promising safety profile in polyplex formulation.

The synthetic peptide SVVYGLR promotes cell motility of myogenic cells and facilitates differentiation in skeletal muscle regeneration.

The present study was designed to evaluate the effects of the osteopontin-derived multifunctional short peptide, SVVYGLR (SV) peptide on the biological properties of skeletal muscle-specific myogenic cells. We employed human-derived satellite cells (HSkMSC) and skeletal muscle myoblasts (HSMM) and performed a series of biochemical experiments. The synthetic SV peptide showed no influence on the proliferation and adhesion properties of HSkMSC and HSMM, while it showed a significant increase in cell motility, including migration activities upon treatment with the SV peptide. In a rat model with volumetric loss of masticatory muscle, immunohistochemical staining of regenerating muscle tissue immediately after injury demonstrated an increase of the number of both MyoD- and myogenin-positive cells in SV peptide-treated group. These results suggest that SV peptide plays a potent role in facilitating skeletal muscle regeneration by promoting the migration, and differentiation of myogenic precursor and progenitor cells.

Infection microenvironment-responsive multifunctional peptide coated gold nanorods for bimodal antibacterial applications

The abuse of antibiotics has led to bacterial drug resistance, which hinders traditional treatments for infection. It is a vitally important task to develop new methods for combating bacterial infection. In this work, we design a multifunctional peptide (BrEK) that consists of three functional building blocks: an antimicrobial sequence (BF2b), a gelatinase B responsive linker, and a zwitterionic antifouling block. The BrEK peptide is conjugated onto gold nanorods (AuNRs) via a cysteine on one end of the peptide sequence, with excellent photothermal conversion ability under near-infrared (NIR) light. The obtained AuNR@BrEK are tested in vitro and in vivo for antibiotic capacity against methicillin-resistant Staphylococcus aureus (MRSA). The multifunctional peptide coating provides the nanocomposite excellent biocompatibility, relative long circulation in blood, infection microenvironment responsive targeting ability, and unique antibacterial performance. Ultimately, we aim to eliminate drug resistance bacteria by (1) integrating infection microenvironment responsiveness for efficient targeting, (2) capitalizing on antibacterial capacity of BF2b peptides, and (3) developing photothermal sterilization. Transmittance electron microscopy, dynamic light scatter, and zeta potential measurements are introduced to characterize the synthesis of AuNR@BrEK. The evaluation of particle stability is conducted by Fourier-transform infrared spectroscopy and UV–vis spectroscopy. Photothermal conversion capacity of AuNR@BrEK is examined via photothermal effect measurement. Cell viability and hemolysis assay are conducted to further assess the biocompatibility of AuNR@BrEK (200 μg/mL). Lastly, results from the in vitro and in vivo experiments indicate the outstanding antibacterial activity of AuNR@BrEK (10 mg/kg). Such multifunctional gold nanorods represent an important step forward in the goal to combat bacterial resistance.

Multifunctional Peptide-Conjugated Nanocarriers for Pulp Regeneration in a Full-Length Human Tooth Root

Dental pulp is a highly vascularized tissue, situated in an inextensible environment surrounded by rigid dentinal walls. The pulp receives its blood supply solely from the small apical foramen of a tooth root. Due to the unique anatomy that controls nutrition supply, regeneration of pulp tissue in a full-length tooth root has long been a challenge in regenerative endodontics. In this study, we designed and synthesized a novel multifunctional peptide-conjugated, pH-sensitive, non-viral gene vector for fast revascularization and pulp regeneration in a full-length human tooth root. The multifunctional peptide was designed to have distinctive features, including a cell-penetrating peptide to enhance cellular uptake, a nuclear localization signal peptide to assist in the translocation of an angiogenic gene into the nucleus, and a fluorescent tryptophan residue to visualize and quantify the transfection efficiency. Furthermore, a pH-sensitive dimethylmaleic anhydride (DMA) was integrated with the multifunctional peptide to enhance the transfected gene complex to escape from endosomes/lysosomes after internalization. In vitro experiments showed that the multifunctional non-viral gene vector significantly increased internalization and gene transfection efficiency as well as reduced cytotoxicity. After dental pulp stem cells (DPSCs) were transfected with the multifunctional gene vector/pVEGF complexes, the expression of VEGF from the DPSCs was upregulated for more than eight folds, which in turn greatly enhanced endothelial cell migration and vascular-like tube formation. Six weeks after implantation, the VEGF-transfected DPSCs accelerated new blood vessel formation and the regenerated pulp tissue occupied most of the area in the canal of a full-length human tooth root. The multifunctional peptide conjugated non-viral gene delivery is a safe and effective approach for regenerative endodontics.

Early low-dose ghrelin intervention via miniosmotic pumps could protect against the progressive dopaminergic neuron loss in Parkinson's disease mice

Ghrelin has been identified as a multifunctional peptide that has a potential application for treating Parkinson's disease (PD). The objective of this study was to assess the effects of subcutaneous administration of low-dose ghrelin via miniosmotic pumps on PD progression. The decreased levels of total and active ghrelin in plasma were rescued by ghrelin administration in PD mice. Interestingly, ghrelin did not affect weight gain in wild-type mice but improved weight loss in PD mice. We observed the attenuation of dopaminergic neuron loss in substantia nigra and a low level of dopamine content in the striatum in PD mice with ghrelin treatment. Ghrelin administration could improve the microenvironment of dopaminergic neurons by inhibiting microglial proliferation and proinflammatory cytokine expression and could enhance cell survival by upregulating Bcl-2/Bax ratio and superoxide dismutase1 protein level in the substantia nigra of PD mice. Subcutaneous administration of low-dose ghrelin could prevent the onset of the progression of PD and also provide a possible method for ghrelin application to cure PD.