Tachykinin Neuropeptides and Amyloid ß (25-35) Assembly: Friend or Foe?

Amyloid ß (Aß) protein is responsible for Alzheimer's disease, and one of its important fragments, Aß(25-35), is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aß(25-35)'s toxicity in cells even though they share similar primary structures with Aß(25-35). Here, we seek to understand the molecular mechanisms of how these peptides interact with Aß(25-35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aß(25-35), NK, Kassinin, Substance P, and mixtures of the latter three with Aß(25-35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of Aß(25-35), which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aß(25-35), NK, Kassinin, and Substance P do not form hexamers with a ß-sheet structure like Aß(25-35). The hexamer structure of Aß(25-35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aß(25-35) are discussed.

Radiolabeled Gold Nanoseeds Decorated with Substance P Peptides: Synthesis, Characterization and In Vitro Evaluation in Glioblastoma Cellular Models.

Despite some progress, the overall survival of patients with glioblastoma (GBM) remains extremely poor. In this context, there is a pressing need to develop innovative therapy strategies for GBM, namely those based on nanomedicine approaches. Towards this goal, we have focused on nanoparticles (AuNP-SP and AuNP-SPTyr8) with a small gold core (ca. 4 nm), carrying DOTA chelators and substance P (SP) peptides. These new SP-containing AuNPs were characterized by a variety of analytical techniques, including TEM and DLS measurements and UV-vis and CD spectroscopy, which proved their high in vitro stability and poor tendency to interact with plasma proteins. Their labeling with diagnostic and therapeutic radionuclides was efficiently performed by DOTA complexation with the trivalent radiometals 67Ga and 177Lu or by electrophilic radioiodination with 125I of the tyrosyl residue in AuNP-SPTyr8. Cellular studies of the resulting radiolabeled AuNPs in NKR1-positive GBM cells (U87, T98G and U373) have shown that the presence of the SP peptides has a crucial and positive impact on their internalization by the tumor cells. Consistently, 177Lu-AuNP-SPTyr8 showed more pronounced radiobiological effects in U373 cells when compared with the non-targeted congener 177Lu-AuNP-TDOTA, as assessed by cell viability and clonogenic assays and corroborated by Monte Carlo microdosimetry simulations.

The Regenerative Potential of Substance P.

Wound healing is a highly coordinated process which leads to the repair and regeneration of damaged tissue. Still, numerous diseases such as diabetes, venous insufficiencies or autoimmune diseases could disturb proper wound healing and lead to chronic and non-healing wounds, which are still a great challenge for medicine. For many years, research has been carried out on finding new therapeutics which improve the healing of chronic wounds. One of the most extensively studied active substances that has been widely tested in the treatment of different types of wounds was Substance P (SP). SP is one of the main neuropeptides released by nervous fibers in responses to injury. This review provides a thorough overview of the application of SP in different types of wound models and assesses its efficacy in wound healing.

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional µ/δ-Opioid Agonist-Neurokinin Antagonist Peptidomimetics.

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic µ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual µ/δ opioid agonist H-Dmt-d-Arg-Aba-ßAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range µ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

Substance P containing peptide gene delivery vectors for specifically transfecting glioma cells mediated by a neurokinin-1 receptor.

Gene therapy provides a promising treatment for glioblastoma multiforme, which mainly depends on two key aspects, crossing the blood brain barrier (BBB) effectively and transfecting target cells selectively. In this work, we reported a series of peptide-based vectors for transfecting glioma cells specifically consisting of several functional segments including a cell-penetrating peptide, targeting segment substance P (SP), an endosomal escape segment, a PEG linker and a stearyl moiety. The conformations and DNA-loading capacities of peptide vectors and the self-assembly behaviors of peptide/pGL3 complexes were characterized. The in vitro gene transfection was evaluated in U87, 293T-NK1R, and normal 293T cell lines. The transfection efficiency ratio of P-02 (SP-PEG4-K(C18)-(LLHH)3-R9) to Lipo2000 in the U87 cell line was about 36% higher than that in the 293T cell line. The neurokinin-1 receptor (NK1R) in U87 cells mediated the transfection process via interactions with the ligand SP in peptide vectors. The mechanism of NK1R mediated transfection was demonstrated by the use of gene-modified 293T cells expressing NK1R, as well as the gene transfection in the presence of free SP. Besides, P-02 could promote the pGL3 plasmids to cross the BBB model in vitro and achieved the EGFP gene transfection in the brain of zebrafish successfully. The designed peptide vectors, owing to their specific transfection capacity in glioma cells, provide a potential approach for glioblastoma multiforme gene therapy.

Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments.

Electron-based dissociation (ExD) produces uncluttered mass spectra of intact proteins while preserving labile post-translational modifications. However, technical challenges have limited this option to only a few high-end mass spectrometers. We have developed an efficient ExD cell that can be retrofitted in less than an hour into current LC/Q-TOF instruments. Supporting software has been developed to acquire, process, and annotate peptide and protein ExD fragmentation spectra. In addition to producing complementary fragmentation, ExD spectra enable many isobaric leucine/isoleucine and isoaspartate/aspartate pairs to be distinguished by side-chain fragmentation. The ExD cell preserves phosphorylation and glycosylation modifications. It also fragments longer peptides more efficiently to reveal signaling cross-talk between multiple post-translational modifications on the same protein chain and cleaves disulfide bonds in cystine knotted proteins and intact antibodies. The ability of the ExD cell to combine collisional activation with electron fragmentation enables more complete sequence coverage by disrupting intramolecular electrostatic interactions that can hold fragments of large peptides and proteins together. These enhanced capabilities made possible by the ExD cell expand the size of peptides and proteins that can be analyzed as well as the analytical certainty of characterizing their post-translational modifications.

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.

Mass spectrometric studies on the peptide integrity of substance P and related human tachykinins in human biofluids.

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Electroacupuncture alleviates inflammatory pain via adenosine suppression and its mediated substance P expression.

BACKGROUND: Acupuncture has been widely used for alleviating pain. However, its mechanisms remain largely enigmatic. OBJECTIVE: In the present study, we focused on whether the analgesic effect of electroacupuncture is related to its regulation on adenosine and substance P expression. METHODS: We established chronic inflammatory pain model in rats through a single injection of Complete Freund's Adjuvant, and then we treated animals using daily electroacupuncture. We applied seven bilateral sessions of electroacupuncture (ST36 and BL60, 0.5 to 1.5 mA, initial strength of 0.5 mA, increased by 0.5 mA every 10 minutes, for 30 minutes per session, one section per day) to Complete Freund's Adjuvant rats for seven days. The analgesic effect of electroacupuncture was evaluated by measuring paw withdrawal threshold in rats that received mechanical and thermal stimulation. RESULTS: Daily electroacupuncture stimulation effectively increased paw withdrawal threshold in Complete Freund's Adjuvant rats. Electroacupuncture increased the adenosine level in zusanli. A further study showed that electroacupuncture could decrease substance P, neurokinin-1 receptor, tumor necrosis factor-alpha, interleukin-1ß, interleukin-6 and CD68 levels in dorsal root ganglion. Interestingly, direct injection of adenosine A1 or substance P receptor antagonists, or dorsal nerve root transection could significantly impair electroacupuncture induced analgesic actions in Complete Freund's Adjuvant rats could and reduce the levels of substance P, neurokinin-1 receptor, tumor necrosis factor-alpha, interleukin-1ß, interleukin-6 and CD68. Finally, we confirmed that direct injection of adenosine A1 receptor agonist replicated the analgesic effect of electroacupuncture. CONCLUSION: Our results indicate regulation of adenosine-mediated substance P secretion. Substance P-mediated pathway may be involved in the analgesia process by electroacupuncture in rats.

Electroacupuncture alleviates inflammatory pain via adenosine suppression and its mediated substance P expression / A eletroacupuntura alivia a dor inflamatória por meio da supressão de adenosina e da expressão mediada de substância P

ABSTRACT Background: Acupuncture has been widely used for alleviating pain. However, its mechanisms remain largely enigmatic. Objective: In the present study, we focused on whether the analgesic effect of electroacupuncture is related to its regulation on adenosine and substance P expression. Methods: We established chronic inflammatory pain model in rats through a single injection of Complete Freund's Adjuvant, and then we treated animals using daily electroacupuncture. We applied seven bilateral sessions of electroacupuncture (ST36 and BL60, 0.5 to 1.5 mA, initial strength of 0.5 mA, increased by 0.5 mA every 10 minutes, for 30 minutes per session, one section per day) to Complete Freund's Adjuvant rats for seven days. The analgesic effect of electroacupuncture was evaluated by measuring paw withdrawal threshold in rats that received mechanical and thermal stimulation. Results: Daily electroacupuncture stimulation effectively increased paw withdrawal threshold in Complete Freund's Adjuvant rats. Electroacupuncture increased the adenosine level in zusanli. A further study showed that electroacupuncture could decrease substance P, neurokinin-1 receptor, tumor necrosis factor-alpha, interleukin-1β, interleukin-6 and CD68 levels in dorsal root ganglion. Interestingly, direct injection of adenosine A1 or substance P receptor antagonists, or dorsal nerve root transection could significantly impair electroacupuncture induced analgesic actions in Complete Freund's Adjuvant rats could and reduce the levels of substance P, neurokinin-1 receptor, tumor necrosis factor-alpha, interleukin-1β, interleukin-6 and CD68. Finally, we confirmed that direct injection of adenosine A1 receptor agonist replicated the analgesic effect of electroacupuncture. Conclusion: Our results indicate regulation of adenosine-mediated substance P secretion. Substance P-mediated pathway may be involved in the analgesia process by electroacupuncture in rats.

RESUMO Introdução: A acupuntura tem sido amplamente utilizada para alívio de dor. No entanto, seus mecanismos são muito pouco conhecidos. Objetivo: Investigar a relação entre o efeito analgésico da eletroacupuntura e a regulação da expressão de adenosina e de substância P. Métodos: Utilizou-se um modelo de dor inflamatória crônica em ratos por injeção única do Adjuvante Completo de Freund e, em seguida, os animais foram tratados com eletroacupuntura diariamente. Foram aplicadas sete sessões bilaterais de eletroacupuntura (ST36 e BL60, 0,5 a 1,5 mA, força inicial de 0,5 mA, aumentada em 0,5 mA a cada 10 minutos, 30 minutos por sessão, uma sessão por dia) em ratos com Adjuvante Completo de Freund, por sete dias. O efeito analgésico da eletroacupuntura foi avaliado pela medida do limiar de retirada da pata em ratos que receberam estimulações mecânica e térmica. Resultados: A estimulação diária com eletroacupuntura aumentou efetivamente o limiar de retirada da pata em ratos com Adjuvante Completo de Freund. A eletroacupuntura aumentou o nível de adenosina na região zusanli. Estudos posteriores mostraram que a eletroacupuntura poderia diminuir os níveis de substância P, receptor de neurocinina-1, fator de necrose tumoral-alpha, interleucina-1β, interleucina-6 e CD68 nos gânglios da raiz dorsal. Curiosamente, a injeção direta de antagonistas do receptor de adenosina A1 ou de substância P, ou a transecção da raiz do nervo dorsal, podem prejudicar significativamente as ações analgésicas induzidas pela eletroacupuntura em ratos com Adjuvante Completo de Freund e reduzir os níveis de substância P, receptor de neurocinina-1, fator de necrose tumoral-alfa, interleucina-1β, interleucina-6 e CD68. Por fim, confirmamos que a injeção direta de um agonista do receptor da adenosina A1 reproduziu os efeitos analgésicos da eletroacupuntura. Conclusão: Nossos resultados indicam a regulação da secreção da substância P mediada pela adenosina. A via mediada pela substância P pode estar envolvida no processo de analgesia por eletroacupuntura em ratos.

Improving NK1R-targeted gene delivery of stearyl-antimicrobial peptide CAMEL by conjugating it with substance P.

Specificity is a crucial condition that hampers the application of non-viral vectors for cancer gene therapy. In a previous study, we developed an efficient gene vector, stearyl-CAMEL, using N-terminal stearylation of the antimicrobial peptide CAMEL. Substance P (SP), an 11-residue neuropeptide, rapidly enters cells after binding to the neurokinin-1 receptor (NK1R), which is expressed in many cancer cell lines. In this study, the NK1R-targeted gene vector stearyl-CMSP was constructed by conjugating SP to the C-terminus of stearyl-CAMEL. Our results indicated that stearyl-CMSP displayed significant transfection specificity for NK1R-expressing cells compared with that shown by stearyl-CAMEL. Accordingly, the stearyl-CMSP/p53 plasmid complexes had significantly higher antiproliferative activity against HEK293-NK1R cells than they did against HEK293 cells, while the stearyl-CAMEL/p53 plasmid complexes did not show this specificity in antiproliferative activity. Consequently, conjugation of the NK1R-targeted ligand SP is a simple and successful strategy to construct efficient cancer-targeted non-viral gene vectors.

Charge Retention/Charge Depletion in ESI-MS: Experimental Evidence.

The effects of liquid and gas phase additives (chemical modifiers) on the ion signal distribution for Substance P (SP), recorded with a nanoelectrospray setup, are evaluated. Depletion of the higher charge state of Substance P ([SP+3H]3+) is observed with polar protic gas phase modifiers. This is attributed to their ability to form larger hydrogen-bonded clusters, whose proton affinity increases with cluster size. These clusters are able to deprotonate the higher charge state. "Supercharging agents" (SCAs) as well as aprotic polar gas phase modifiers, which promote the retention of the higher charge state of Substance P, do not form such large clusters under the given conditions and are therefore not able to deprotonate Substance P. Both SCAs and aprotic modifiers form clusters with the higher charge state, leading to stabilization of the charge. Whereas supercharging agents have low vapor pressures and are therefore enriched in late-stage electrospray droplets, the gas phase modifiers are volatile organic solvents. Collision induced dissociation experiments revealed that the addition of a modifier significantly delays the droplet evaporation and ion release process. This indicates that the droplet takes up the gas phase modifier to a certain extent (accommodation). Depending on the modifier's properties either charge depletion or retention may eventually be promoted.

Enhanced cell selectivity of hybrid peptides with potential antimicrobial activity and immunomodulatory effect.

BACKGROUND: Hybridization is a useful strategy to bond the advantages of different peptides into novel constructions. We designed a series of AMPs based on the structures of a synthetic AMP KFA3 and a naturally-occurred host defense peptide substance P (SP) to obtain peptides retaining the high antibacterial activity of KFA3 and the immunomodulatory activity and low cytotoxicity of SP. METHODS: Two repeats of KFA and different C terminal fragments of SP were hybridized, generating a series of novel AMPs (KFSP1-8). The antibacterial activities, host cell toxicity and immunomodulation were measured. The antibacterial mechanisms were investigated. RESULTS: Hybrid peptides KFSP1-4 exerted substantial antibacterial activities against Gram-negative bacteria of standard strains and clinical drug-resistant isolates including E.coli, A.baumannii and P.aeruginosa, while showing little toxicity towards host cells. Compared with KFA3, moderate reduction in α-helix content and the interruption in α-helix continuality were indicated in CD spectra analysis and secondary-structure simulation in these peptides. Membrane permeabilization combined with time-kill studies and FITC-labeled imaging, indicated a selective membrane interaction of KFSP1 with bacteria cell membranes. By specially activating NK1 receptor, the hybrid peptides kept the ability of SP to induce intracellular calcium release and ERK1/2 phosphorylation, but unable to stimulate NF-κB phosphorylation. KFSP1 facilitated the survival of mouse macrophage RAW264.7, directly interacting with LPS and inhibiting the LPS-induced NF-κB phosphorylation and TNF-α expression. CONCLUSION: Hybridization is a useful strategy to bond the advantages of different peptides. KFSP1 and its analogs are worth of advanced efforts to explore their potential applications as novel antimicrobial agents.

Substance P-embedded multilayer on titanium substrates promotes local osseointegration via MSC recruitment.

In this study, the chemokine substance P (SP) was inserted into multilayered systems on titanium (Ti)-based substrates for endogenous mesenchymal stem cell (MSC) recruitment to facilitate bone healing. The multilayer was constructed with cationic chitosan (Chi), SP and anionic gelatin (Gel) via a spin-coater-assisted layer-by-layer (LBL) approach. The characterization results demonstrated that the multilayer system was successfully constructed and was capable of continuously releasing SP for almost 2 weeks. We further confirmed that MSCs grown on SP-modified Ti-based substrates showed improved migration capabilities as well as enhanced secretion of matrix metalloproteinases (MMP2, MMP9), rather than enhanced MSC proliferation and differentiation in vitro. In the CD29+/CD90+ double immunofluorescence assay, the Ti/LBL-SP group showed the highest number of MSCs migrating to the peri-implant area after implantation. Consistently, the Ti/LBL-SP implants also significantly enhanced new bone formation according to the results of micro-CT scanning analysis, H&E staining, Masson's trichrome staining and immunohistochemical staining. The obtained results reveal that SP-modified Ti-based substrates were beneficial for bone formation via recruiting endogenous MSCs.

Labeled substance P as a neuropeptide reporter substance for enzyme activity.

Recently, we developed a bradykinin reporter assay and demonstrated the differing protease activity in Complex Regional Pain Syndrome patients vs. controls. In order to further characterize CRPS pathophysiology, the neuropeptide substance P was evaluated as possible reporter substance, here. It was labeled with a chromophore at the lysine residue and generated two major fragments following incubation with serum (amino acid residues 3-8 and 3-11) which were reproducibly separated by thin-layer chromatography. Dabsylated substance P was shown to be a substrate of angiotensin-converting enzyme. The combination of both bradykinin and substance P reporter substances with specific enzyme inhibitors will shed more light on biochemical pathways in inflammatory processes and pain. Comparative clinical studies are now needed to define the application range of both assays in more detail.

A biuret-derived, MS-cleavable cross-linking reagent for protein structural analysis: A proof-of-principle study.

Chemical cross-linking combined with mass spectrometry (XL-MS) and computational modeling has evolved as an alternative method to derive protein 3D structures and to map protein interaction networks. Special focus has been laid recently on the development and application of cross-linkers that are cleavable by collisional activation as they yield distinct signatures in tandem mass spectra. Building on our experiences with cross-linkers containing an MS-labile urea group, we now present the biuret-based, CID-MS/MS-cleavable cross-linker imidodicarbonyl diimidazole (IDDI) and demonstrate its applicability for protein cross-linking studies based on the four model peptides angiotensin II, MRFA, substance P, and thymopentin.

The effect of Substance P/Heparin conjugated PLCL polymer coating of bioinert ePTFE vascular grafts on the recruitment of both ECs and SMCs for accelerated regeneration.

Artificial vascular grafts consisting of ePTFE have been mainly used in clinics for the treatment of cardiovascular disease. However, artificial grafts can become clogged after a long time due to thrombosis, as graft maturation by endothelialization is limited. The strategy introduced in this study is to induce graft remodeling through interaction between the bioinert graft and the body. The Substance P (SP) and heparin were covalently conjugated with PLCL, an elastic biocompatible copolymer and the Substance P-conjugated PLCL (SP-PLCL) and/or heparin-conjugated PLCL (Hep-PLCL) were vacuum-coated onto ePTFE vascular grafts. To assess the effectiveness of the coating, coated samples were evaluated by implanting them subcutaneously into SD-Rats. Coatings allow grafts to be remodeled by creating a microenvironment where cells can grow by infiltrating into the grafts while also greatly enhancing angiogenesis. In particular, a double coating of Hep-PLCL and SP-PLCL (Hep/SP-PLCL) at four weeks showed markedly improved vascular remodeling through the recruitment of mesenchymal stem cells (MSCs), vascular cells (ECs, SMCs) and M2 macrophages. Based on these results, it is expected that when the Hep/SP-PLCL-coated ePTFE vascular grafts are implanted in situ, long-term patency will be assured due to the appropriate formation of an endothelial layer and smooth muscle cells in the grafts like native vessels.

Substance P-loaded electrospun small intestinal submucosa/poly(ε-caprolactone)-ran-poly(l-lactide) sheet to facilitate wound healing through MSC recruitment.

In this work, we prepared an electrospun small intestinal submucosa/poly(ε-caprolactone)-ran-poly(l-lactide) (SIS/PCLA) sheet onto which substance P (SP) was loaded, and this was employed as a cell-free scaffold for wound healing through the mobilization of human mesenchymal stem cells (hMSCs). SP release from the SP-loaded scaffold was 42% at 12 h and 51% at 24 h due to an initial burst of SP, but after 1 day, it exhibited a linear release profile and was released at a sustained rate for 21 days. The SP-loaded SIS/PCLA sheet exhibited higher in vitro and in vivo hMSC migration than did the PCLA and SIS/PCLA sheets. Large hMSCs injected into the tail vein of mice models migrated towards the wound to a greater extent in the presence of the SP-loaded SIS/PCLA sheet than with the PCLA and SIS/PCLA sheets, as confirmed by the CD44 and CD29 markers of recruited hMSCs. In animal wound models, significantly higher wound contraction (∼97%) in the group treated with the SP-loaded SIS/PCLA sheet was observed compared with the PCLA (∼74%) and SIS/PCLA (∼84%) groups at 3 weeks. In addition, SP-loaded SIS/PCLA-treated animals showed significant epidermal regeneration and collagen density (56%) in the mature granulation tissue at 3 weeks compared to the PCLA and SIS/PCLA groups. The wound area after SP-loaded SIS/PCLA sheet treatment also showed high blood vessel formation at the early stage, resulting in enhanced wound healing. Furthermore, the SP-loaded SIS/PCLA group exhibited a lower macrophage count (2.9%) than did the PCLA (7.7%) and SIS/PCLA (3.4%) groups. It was thus confirmed that the use of SP-loaded SIS/PCLA sheet as a cell-free scaffold could effectively enhance wound healing through MSC recruitment.

Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y.

Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen.

Structural analysis of the neuropeptide substance P by using vibrational spectroscopy.

Substance P (SP) is one of the most studied peptide hormones and knowing the relationship between its structure and function may have important therapeutic applications in the treatment of a variety of stress-related illnesses. In order to obtain a deeper insight into its folding, the effects of different factors, such as pH changes, the presence of Ca2+ ions, and the substitution of the Met-NH2 moiety in the SP structure, was studied by Raman and infrared spectroscopies. SP has a pH-dependent structure. Under acidic-neutral conditions, SP possesses a prevalent ß-sheet structure although also other secondary structure elements are present. By increasing pH, a higher orderliness in the SP secondary structure is induced, as well as the formation of strongly bound intermolecular ß-strands with a parallel alignment, which favour the self-assembly of SP in ß-aggregates. The substitution of the Met-NH2 moiety with the acidic functional group in the SP sequence, giving rise to a not biologically active SP analogue, results in a more disordered folding, where the predominant contribution comes from a random coil. Conversely, the presence of Ca2+ ions affects slightly but sensitively the folding of the polypeptide chain, by favouring the α-helical content and a different alignment of ß-strands; these are structural elements, which may favour the SP biological activity. In addition, the capability of SERS spectroscopy to detect SP in its biologically active form was also tested by using different metal nanoparticles. Thanks to the use of silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride, SP can be detected at very low peptide concentration (~ 90 nM). However, the SERS spectra cannot be obtained under alkaline conditions since both the formation of SP aggregates and the lack of ion pairs do not allow a strong enough interaction of SP with silver NPs. Graphical abstract.