Acid Peptide Research Articles

Selective delivery of G-quadruplex ligand in glioma cell lines: the power of cyclic-RGD peptide

Compounds targeting non-canonical secondary structures of nucleic acids, known as G-quadruplexes, are highly cytotoxic, both for cancer and healthy cells, because of their action mechanism’s lack of appropriate selectivity. The targeted delivery of cytotoxic molecules to cancer cells is a valuable strategy to expand the repertoire of potential drugs, especially for cancer types for which new therapeutic tools are urgently needed, like glioblastoma. In this work, we conjugated a cyclic arginyl-glycyl-aspartic acid peptide to a naphthalene diimide, previously described as a highly performing stabilizing ligand for DNA G-quadruplexes, to specifically target glioma cells overexpressing RGD-binding integrin receptors. Our results, including confocal microscopy and cell toxicity assays, demonstrated improved efficacy and selective cellular absorption of the new conjugate without affecting the NDI’s ability to interact with the G4 target.

A novel antifungal peptide, SP1.2, from Rhodopseudomonas palustris against the rice blast pathogen.

Rice blast has a significant detrimental impact on rice yields, so developing efficient biological control technologies is an effective means for rice blast prevention and control. The GroEL protein has proven to be effective at preventing and managing the pathogenicity of rice blast. Here, we analyzed the amino acid sequence of the GroEL protein and synthesized the '60 kDa chaperonin signature' (350-373 amino acids) peptide SP1.2, which has potent antifungal activity. Notably, the SP1.2 peptide exhibited potent fungicidal activity against Magnaporthe oryzae, effectively inhibiting appressorium germination. Electron microscopy revealed that SP1.2 disrupted the fungal plasma membrane and bound to multiple bioactive phosphoinositides in vitro, triggering the production of reactive oxygen species. Furthermore, it also caused an increase in the acetylation of M. oryzae and induced autophagy in cells. The spray application of SP1.2 significantly reduced the number of disease spots caused by the fungal pathogen M. oryzae in rice, enhancing the defense response of rice plants. Field trials showed that the control effect was 64.59% after spraying SP1.2. Our study illustrates the antifungal activity of the structurally unique SP1.2 peptide against plant fungal pathogens and paves the way for the future development of this class of peptides as antifungal agents. © 2024 Society of Chemical Industry.

STING agonists and PI3Kγ inhibitor co-loaded ferric ion-punicalagin networks for comprehensive cancer therapy

Nanoparticles-based drug delivery system has been a promising approach for the treatment of colorectal cancer (CRC), which can be combined with chemotherapy, targeted therapy and immunotherapy to improve the treatment of CRC. 2′3’ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is an agonist of the STING signaling pathway activating antitumor immunity. IPI-549 is a small-molecule inhibitor for phosphatidylinositol 3-kinase γ (PI3Kγ), which can induce M1 macrophages polarization to provide pro-inflammatory microenvironment to suppress tumors. Here, we developed a ferric ion-punicalagin network (Fe-PU), which can be not only used as an inducer of ferroptosis, but also serve as a carrier to load cGAMP and IPI-549 to obtain nanohybrid (Fe-PU/CD-IPI). In order to improve the delivery effect and targeted ability to CRC, a cyclic arginine-glycine-aspartic acid peptide linked-bovine serum albumin were utilized to modify Fe-PU/CD-IPI to prepare nanohybrid Fe-PU/CD-IPI@cBSA. The therapeutic effect of various nanohybrids were validated in the mice with spontaneous tumor in the colorectal area and tumor-bearing mice, which lead to the increase of ferroptosis, the activation of STING signaling pathway, and the repolarization of macrophages. Collectively, the cGAMP and IPI-549 co-loaded nanohybrids effectively reshaped the tumor immune microenvironment, and exhibited prominent treatment effect of anti-colorectal cancer in vitro, patient-derived organoids, and in vivo.

Synthesis and Biological Evaluation of Amino Acid and Peptide Conjugates of 5-Bromovaleric Acid.

Among various carboxylic acid derivatives, valeric acid or pentanoic acid is found to be widely distributed in nature. It is a straight-chain alkyl carboxylic acid containing five carbon atoms. Due to the therapeutic value of valeric acid, it is used as a versatile nucleus in the pharmaceutical field. Valeric acid derivatives are associated with a broad spectrum of biological activities, like anticonvulsant, antiplatelet, antidiabetic, and plant growth activities. It has previously been revealed that peptide derivatives of carboxylic acids are accountable for enhanced antimicrobial activity. Therefore, it was hypothesized that coupling peptides with valeric acid would increase the antimicrobial properties of the target compounds. So, the objective of the present study was to synthesize peptide derivatives of 5-bromovaleric acid and evaluate their antibacterial and antifungal activities. 5-bromovaleric acid was synthesized by the reaction of cyclopentanone and hydrogen peroxide in the presence of copper bromide and sodium bromide. Additionally, 5-bromovaleric acid was coupled with amino acid methyl esters, dipeptides, tripeptides, and tetrapeptides in the presence of dicyclohexylcarbodimide (DCC) and N-methylmorpholine (NMM) as a base under continuous stirring for 36 hours to produce its peptide derivatives. The results obtained showed that 5-bromovaleric acid possesses more potent antibacterial activity than N-terminal 5-bromovaleric acid conjugates of selected di-, tri, and tetra peptide Cterminal methyl esters against ciprofloxacin as a standard. The selected dipeptide and tripeptide Nterminal 5-bromovaleric acid-conjugated C-terminal methyl ester derivatives were more active than the selected tetrapeptide methyl ester analogue. Using fluconazole as a reference, the antifungal efficacy of 5-bromovaleric acid against C. albicans and A. niger declined as it was combined with C-terminal methyl esters of selected dipeptides, tripeptides, and tetrapeptides. The novel selected peptide derivatives had less antibacterial and antifungal action than the parent 5-bromovaleric acid. Antibacterial and antifungal investigations showed that 5- bromopentanoic acid peptide derivatives might impair antimicrobial efficacy. Further, attaching 5- bromopentanoic acid to di, tri, and tetra peptides did not boost their antibacterial potential.

Non-invasively differentiate non-alcoholic steatohepatitis by visualizing hepatic integrin αvβ3 expression with a targeted molecular imaging modality

BACKGROUND Non-invasive methods to diagnose non-alcoholic steatohepatitis (NASH), an inflammatory subtype of non-alcoholic fatty liver disease (NAFLD), are currently unavailable. AIM To develop an integrin αvβ3-targeted molecular imaging modality to differentiate NASH. METHODS Integrin αvβ3 expression was assessed in Human LO2 hepatocytes Scultured with palmitic and oleic acids (FFA). Hepatic integrin αvβ3 expression was analyzed in rabbits fed a high-fat diet (HFD) and in rats fed a high-fat, high-carbohydrate diet (HFCD). After synthesis, cyclic arginine-glycine-aspartic acid peptide (cRGD) was labeled with gadolinium (Gd) and used as a contrast agent in magnetic resonance imaging (MRI) performed on mice fed with HFCD. RESULTS Integrin αvβ3 was markedly expressed on FFA-cultured hepatocytes, unlike the control hepatocytes. Hepatic integrin αvβ3 expression significantly increased in both HFD-fed rabbits and HFCD-fed rats as simple fatty liver (FL) progressed to steatohepatitis. The distribution of integrin αvβ3 in the liver of NASH cases largely overlapped with albumin-positive staining areas. In comparison to mice with simple FL, the relative liver MRI-T1 signal value at 60 minutes post-injection of Gd-labeled cRGD was significantly increased in mice with steatohepatitis (P < 0.05), showing a positive correlation with the NAFLD activity score (r = 0.945; P < 0.01). Hepatic integrin αvβ3 expression was significantly upregulated during NASH development, with hepatocytes being the primary cells expressing integrin αvβ3. CONCLUSION After using Gd-labeled cRGD as a tracer, NASH was successfully distinguished by visualizing hepatic integrin αvβ3 expression with MRI.

Anti‑angiogenic effect of Bryopsis plumosa‑derived peptide via aquaporin 3 in non‑small cell lung cancer.

Developing novel anti‑angiogenic agents with minimal toxicity is notably challenging for cancer therapeutics. The discovery and development of peptides, whether derived from natural sources or synthesized, has potential for developing anti‑angiogenic agents characterized by their ability to penetrate cancer cells, high specificity and low toxicity. The present study identified a Bryopsis plumose‑derived anticancer and anti‑angiogenesis marine‑derived peptide 06 (MP06). A 22‑amino acid peptide was synthesized and conjugated with fluorescein isothiocyanate (FITC‑MP06) for intracellular localization in H1299 non‑small cell lung cancer cells. Regulatory effects of this peptide on the viability, migration and self‑renewal of lung cancer cells was assessed. Furthermore, anti‑angiogenic effect of MP06 was investigated by monitoring vascular tube formation in human umbilical vein endothelial cells and a zebrafish model. Aquaporin (AQP)3, a membrane channel in various tissues, is involved in regulating stemness, epithelial‑mesenchymal transition (EMT) and angiogenesis. MP06 downregulated AQP3 expression. Consistently, AQP3 knockdown by RNA silencing downregulated its gene expression, leading to a decrease in stemness, EMT and angiogenesis properties in H1299 cells. MP06 could thus serve as a novel therapeutic target with anticancer and angiogenesis properties for non‑small cell lung cancer.

Efficient Neutralizing Antibodies Induction by Human Parvovirus B19 Epitope-Presenting Protein Nanoparticles.

Human parvovirus B19 (B19V) causes fetal hydrops in pregnant women. Despite the significant impact of this virus, effective vaccines remain unclear. In this study, we successfully engineered B19V protein nanoparticles by fusing the N-terminal receptor-binding domain corresponding to 5-80 amino acids of VP1 with two distinct types of self-assembling protein nanoparticles. Gel filtration and electron microscopic analysis confirmed the spherical assembly of the antigen-fused nanoparticles. The purified nanoparticles are efficiently bound to the surface of UT7/Epo-S1 cells, which are semi-permissive hosts for B19V infection. Immunization of BALB/c mice with VP1u 5-80 nanoparticles elicited a robust production of B19V-specific IgG antibodies compared to single VP1u 5-80 peptides. Moreover, a neutralization assay using B19V derived from a blood donor sample revealed that antibodies from mice immunized with VP1u 5-80 nanoparticles exhibited stronger infection-neutralizing activity. These findings suggest that nanoparticle formation plays a crucial role in enhancing the immunogenicity of the B19V VP1u 5-80 amino acid peptide and that these nanoparticles could serve as promising vaccine candidates, effectively inducing immunity against B19V.

Abstract 4136614: The design of novel therapeutics that target the L-type calcium channel to prevent hypertrophic cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is an inherited autosomal dominant disease of the sarcomere. Pathogenic features include ventricular hypertrophy, increased myofilament calcium sensitivity, myocardial fibrosis, and diastolic dysfunction. At the level of the myocyte there is cytoskeletal disarray, hypercontractility and altered mitochondrial function. Mitochondrial dysfunction is considered to be a key driver in HCM pathology. Research question: We previously demonstrated that the L-type Ca 2+ channel plays a role in the development of HCM facilitated by a structural-functional communication with mitochondria that can be regulated via the alpha interaction domain (AID) of the channel. In search of a preventative HCM therapy, we explored the efficacy of amino acid peptide variants that correspond to the AID of the cardiac L-type Ca 2+ channel. Methods and Results: Consistent with in silico predictions , competition binding assays confirmed that 4 variant peptides bound with higher affinity to the beta subunit than the AID peptide. In vitro studies confirmed that 3 of the 4 peptides could decrease the characteristic hypermetabolic state in myocytes isolated from a murine model of human HCM ( cTnI-G203S ). In vivo treatment of cTnI-G203S mice with peptide variants prevented the development of HCM and the development of fibrosis, in the absence of alterations in blood pressure, or kidney and liver function or changes in behaviour. Of note, the peptide variants also significantly improved contractile function. Similar effects were measured in αMHC 403/+ mice expressing the MYH6 mutation. Conclusions: Here we describe a first in class therapy that uniquely targets the L-type Ca 2+ channel to modify mitochondrial function and prevent hypertrophic cardiomyopathy. The peptides may be effective for treatment of HCM broadly because the mechanism of action involves the modification of mitochondrial function and impaired energy metabolism that is a common characteristic and driver of the pathology.

Liquid Crystal Behavior of Uniform Short Rods Made from Computationally Designed Parallel Coiled Coil Building Blocks.

Parallel, homotetrameric coiled coils were computationally designed using 29 amino acid peptides. These parallel coiled coils, called "bundlemers", have C2 symmetry, with all N-termini displayed from one end of the nanoparticle and all C-termini from the opposite end. This anisotropic display of the peptide termini allowed for the functionalization of two sets of nanoparticles with either maleimide or thiol functionality at the N-terminal region of the constituent peptides. The thiol-Michael conjugation reaction between the N-terminal end of complementary bundlemer nanoparticles formed monodisperse, rigid bundlemer dimer, called "dibundlemer", rods. The constituent, individual bundlemer nanoparticles were characterized with small-angle X-ray scattering (SAXS), Förster resonance energy transfer (FRET), and circular dichroism (CD) spectroscopy to confirm the parallel assembly of the coiled coils, consistent with the computational design. The dibundlemer rods were characterized with SAXS to reveal the uniform dibundlemer nature of the rods. Optical birefringence is observed in concentrated samples of the rods, with polarized optical microscopy (POM) revealing a nematic liquid crystalline behavior.

Biosensor for integrin inhibition of mammalian cell adhesion and migration using micropatterned cell culture substrate and retroreflective optical signaling

Integrins are a family of transmembrane receptors that play a crucial role in cell adhesion and migration. Integrins can uniquely transduce biochemical signals bidirectionally across the membrane and physically link the cell-cell and cell-extracellular matrix (ECM) with ligand bonds. The arginyl-glycyl-aspartic acid (RGD) peptide motif is present in the ECM as a minimal recognition sequence for integrins. To leverage this property in cell-based therapy, RGD variants, such as cyclic-type RGDfK (c(RGDfK)), which share a similar structure with RGD but exhibit a higher affinity for integrins, have been developed. However, because most evaluation methods for newly developed RGD variants focus on affinity strength, tools for cellular effects are required. In this study, we developed a new platform that integrates micropatterned three-dimensional cell culture substrates with a non-spectroscopic optical analysis system to quantitatively analyze the effects of RGD variants on cell adhesion and migration. The specially micropatterned substrate provides a cell adhesive and migration area to provide a restricted analysis area. Owing to the characteristics of retroreflective Janus particles (RJPs), a non-spectroscopic optical analysis system provides long-term stable optical verification properties and a simple optical setup. These techniques were integrated to quantitatively determine the integrin inhibitory effect of various concentrations of RGD variant. To demonstrate the efficacy of the developed cellular level RGD variant testing platform, the model cell line L929 fibroblast and model RGD variant c(RGDfK) were analyzed ranging from 0 to 10 μM. The results showed that the developed system could effectively and quantitatively analyze the effects of RGD variants on cells across various concentrations.

Multi-Epitopic Peptide Vaccine Against Newcastle Disease Virus: Molecular Dynamics Simulation and Experimental Validation.

Newcastle disease virus (NDV) is a highly contagious and economically devastating pathogen affecting poultry worldwide, leading to significant losses in the poultry industry. Despite existing vaccines, outbreaks continue to occur, highlighting the need for more effective vaccination strategies. Developing a multi-epitopic peptide vaccine offers a promising approach to enhance protection against NDV. Here, we aimed to design and evaluate a multi-epitopic vaccine against NDV using molecular dynamics (MD) simulation. We retrieved NDV sequences for the fusion (F) protein and hemagglutinin-neuraminidase (HN) protein. Subsequently, B-cell and T-cell epitopes were predicted. The top potential epitopes were utilized to design the vaccine construct, which was subsequently docked against chicken TLR4 and MHC1 receptors to assess the immunological response. The resulting docked complex underwent a 1 microsecond (1000 ns) MD simulation. For experimental evaluation, the vaccine's efficacy was assessed in mice and chickens using a controlled study design, where animals were randomly divided into groups receiving either a local ND vaccine or the peptide vaccine or a control treatment. The 40 amino acid peptide vaccine demonstrated strong binding affinity and stability within the TLR4 and MHC1 receptor-peptide complexes. The root mean square deviation of peptide vaccine and TLR4 receptor showed rapid stabilization after an initial repositioning. The root mean square fluctuation revealed relatively low fluctuations (below 3 Å) for the TLR4 receptor, while the peptide exhibited higher fluctuations. The overall binding energy of the peptide vaccine with TLR4 and MHC1 receptors amounted to -15.7 kcal·mol-1 and -36.8 kcal·mol-1, respectively. For experimental evaluations in mice and chicken, the peptide vaccine was synthesized using services of GeneScript Biotech® (Singapore) PTE Limited. Experimental evaluations showed a significant immune response in both mice and chickens, with the vaccine eliciting robust antibody production, as evidenced by increasing HI titers over time. Statistical analysis was performed using an independent t-test with Type-II error to compare the groups, calculating the p-values to determine the significance of the immune response between different groups. Multi-epitopic peptide vaccine has demonstrated a good immunological response in natural hosts.

Histone Deacetylase 7-Derived 7-Amino Acid Peptide Increases Skin Wound Healing via Regulating Epidermal Fibroblast Proliferation and Migration.

Due to the complexity of wound healing, how to achieve successful healing is a significant clinical challenge. In this study, we found that the histone deacetylase-7-derived 7-amino acid peptide (7A, MHSPGAD), especially its phosphorylated version 7Ap (MH[pSer]PGAD), increased dermal fibroblast cell HDFα proliferation and migration via elevated delta-catenin (CTNND1) serine phosphorylation-mediated beta-catenin (CTNNB) nuclear translocation and subsequent upregulation of c-Myc and cyclin D1 expression. 7Ap physically interacted with platelet-derived growth factor receptor (PDGFR) and increased PDGFR interaction with cyclin-dependent kinase 6 (CDK6). The PDGFR siRNA or CDK6 siRNA knockdown ablated 7AP-induced CTNND1 phosphorylation and subsequent c-Myc/cyclin D1 expression, indicating a novel 7Ap-PDGFR-CDK6-CTNND1/CTNNB signal pathway in regulating fibroblast proliferation and migration. Furthermore, 7Ap increased human umbilic vein endothelial cell proliferation and tube formation, suggesting an angiogenic effect. In a full-thickness excision wound rat model, the local administration of 50 ng/mL of 7Ap in hydrogel exerted a similar effect as 1 μg/mL vascular endothelial growth factor on accelerating wound healing, featured by enhanced fibroblast proliferation and migration, collagen deposition, and increased new vessel formation during the early phase of wound healing. Taken together, this study not only elicited a novel signal pathway in fibroblast proliferation but also paved an avenue to develop 7Ap as a treatment option for skin wound healing.

Peptide-Based Rapid and Selective Detection of Mercury in Aqueous Samples with Micro-Volume Glass Capillary Fluorometer.

Mercury, a toxic heavy metal produced through both natural and anthropogenic processes, is found in all of Earth's major systems. Mercury's bioaccumulation characteristics in the human body have a significant impact on the liver, kidneys, brain, and muscles. In order to detect Hg2+ ions, a highly sensitive and specific fluorescent biosensor has been developed using a novel, modified seven amino acid peptide, FY7. The tyrosine ring in the FY7 peptide sequence forms a 2:1 complex with Hg2+ ions that are present in the water-based sample. As a result, the peptide's fluorescence emission decreases with higher concentrations of Hg2+. The FY7 peptide's performance was tested in the presence of Hg2+ ions and other metal ions, revealing its sensitivity and stability despite high concentrations. Conformational changes to the FY7 structure were confirmed by FTIR studies. Simultaneously, we designed a miniaturized setup to support an in-house-developed micro-volume capillary container for volume fluorometry measurements. We compared and verified the results from the micro-volume system with those from the commercial setup. The micro-volume capillary system accommodated only 2.9 µL of sample volume, allowing for rapid, sensitive, and selective detection of toxic mercury (II) ions as low as 0.02 µM.

Molecular determinants of the selectivity and potency of α-conotoxin Vc1.1 for human nicotinic acetylcholine receptors

The α-conotoxins (α-Ctxs) are short, disulfide-rich peptides derived from the venom of the Conus marine snails, primarily acting as antagonists of nicotinic acetylcholine receptors (nAChRs). Specifically, α-Ctx Vc1.1, a 16-amino acid peptide from C. victoriae, competitively antagonizes non-muscle nAChRs, inhibits nicotine-induced currents in bovine chromaffin cells, and alleviates neuropathic pain in rat models. Although Vc1.1 selectively inhibits rat α9α10 nAChRs, its potency and selectivity across human nAChR subtypes remain unresolved. In this study, we assessed the activity of Vc1.1 on human (h) nAChRs heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique and simulated interactions using computational modelling. Vc1.1 selectively antagonized homomeric α9 and heteromeric α3β2 nAChRs, with half-maximal inhibitory concentrations (IC50) of 160 nM and 232 nM, respectively. At hα9[N179A]α10, Vc1.1 exhibited a 20-fold decrease in potency compared to hα9α10, due to the loss of hydrogen bonding with Vc1.1-D11. Conversely, Vc1.1 was four-fold more potent at hα3β2[E86A] compared to hα3β2, possibly influenced by the proximal residue β2-K104, as suggested by molecular dynamics (MD) simulations. Additionally, Vc1.1’s potency doubled at hα9[N213K]α10, whereas it remained unchanged at hα9[N213R]α10 nAChRs. MD simulations indicate that altered interactions between the mutant hα9 N179A, N213K, or N213R side chains and Vc1.1-D5 may partly explain these changes in potency. The inhibitory action of Vc1.1 at α9-containing nAChRs is particularly relevant given their role in neuroinflammation, presenting a potential therapeutic pathway for alleviating neuropathic and inflammatory pain. This study provides valuable insights for the rational design of Vc1.1-derived α-Ctxs with enhanced nAChR subtype selectivity.

Construction of Aryl Azide‐ and Triazole‐Based Unnatural Amino Acid Motifs via the Pd(II)‐Catalyzed C(sp3)−H Arylation

AbstractThis paper reports a method for the synthesis of examples of aryl azide‐ and triazole moiety‐based unnatural amino acid motifs using the Pd(II)‐catalyzed arylation of the prochiral C(sp3)−H bonds of carboxamides of amino acids as one of the key transformations. Synthesis of racemic and enantioenriched aryl azide‐based unnatural amino acid motifs including, norvaline, leucine, norleucine, phenylalanine, 2‐aminobutyric acid, 2‐aminooctanoic acid, and non‐α‐amino acid derivatives were shown. Additionally, we attempted the synthesis of triazole moiety‐installed norvaline, leucine, norleucine, phenylalanine, 2‐aminobutyric acid, 2‐aminooctanoic acid, and non‐α‐amino acid derivatives via the Cu‐catalyzed Click reaction. We also demonstrated the removal of the protecting groups and the synthesis of free amino group‐containing aryl azide‐ or triazole‐based unnatural amino acid motifs and peptides. Generally, azide moiety‐containing unnatural amino acid motifs are important molecular tools in chemical biology. Furthermore, aryl azide‐ and triazole moiety‐containing unnatural amino acid scaffolds are notable substrates in medicinal chemistry and drug discovery research. Accordingly, this work contributes to enriching the library of aryl azide‐ and triazole‐based unnatural amino acid derivatives.

AtheroVax: a peptide vaccine with a therapeutic effect in animal model of chronic inflammation

Abstract Background Inflammation is a driver of atherosclerosis, but inflammatory mediators are essential components of the immune response. AtheroVax targets only pathogenic forms of sTNFR2 while preserving the membrane-bound (wild-type) TNFR2 and its contributions to a non-pathogenic immune response. We hypothesize that the vaccine mediated clearance of sTNRF2 will be more specific and offers long-term treatment options. The aim of this study is to investigate the therapeutic effect of an sTNFR2-targeting peptide (14 amino acid) vaccine, AtheroVax, in a rat model of acetic acid-induced colitis. Methods Two studies were conducted. In the first study 100 male outbred white Wistar rats (age 8 weeks, weight ~250 g) were used to evaluate a subcutaneous (SQ) injections of human sTNFR2 peptide sequence. In the second study 80 rats were used to evaluate SQ injections of the rat sTNFR2 sequence. After the adaptation period, the animals were randomly divided into treatment groups. In study 1, group A (n = 30), phosphate-buffered saline (PBS), group B (n = 30), 25 μg of alum diluted in PBS, group C (n = 30), 25 μg of alum + 25 μg of peptide diluted in PBS, and group D (n = 10) no colitis controls. In study 2, group A (n = 25), phosphate-buffered saline (PBS), group B (n = 25), 25 μg of alum + 25 μg of rat sTNFR2 peptide, group C (n = 30), 25 μg of alum + 75 μg of rat sTNFR2 peptide, and group D (n = 5) no colitis controls. The animals were kept under standard vivarium conditions (temperature 22±2°C, relative humidity 50±5%, light/dark cycles 12 h/12 h). Animals were fed ad libitum standard laboratory chow and water under the supervision of trained personnel. Results The short 14 amino acid peptides administered prior to initiating colitis significantly improved the course of colitis in both studies by significantly (p<0.05) reducing weight loss, fever, bleeding and diarrhea, and disease severity as well as providing a survival benefit (p<0.05) in rats with colitis. AtheroVax also significantly (p<0.001) reduced serum levels of TNF-α and IL-6. Histopathology evaluation reveals preservation of colon cellular architecture in vaccinated animals in both studies. Immunohistochemistry and examination of spleen and lymph nodes provides supporting evidence of the vaccine protection from chronic inflammation. Conclusion Our studies have shown that AtheroVax may be an effective therapeutic candidate for reducing chronic inflammation. These pre-clinical findings will provide a basis to further evaluate this novel vaccine in follow-up pre-clinical studies. A vaccine limiting the progression of atherosclerosis will greatly contribute to the reduction in morbidity and mortality from cardiovascular disease.

Protection of mice against cecal ligation and puncture-induced polymicrobial sepsis by a Fasciola hepatica helminth defence molecule.

Sepsis results from a dysregulated host immune response to infection and is responsible for ~11 million deaths each year. In the laboratory, many aspects of sepsis can be replicated using a cecal ligation and puncture (CLP) model, which is considered the most clinically relevant rodent model of sepsis. In the present study, histological and biomarker multiplex analyses revealed that the CLP model initiated a large-scale inflammatory response in mice by 24 h, with evidence of acute organ damage by 48-72 h. While many typical pro-inflammatory cytokine/chemokines were systemically elevated, a specific array including IL-10, eotaxin, MIP-1α, MIP-1β, MCP-1 and RANTES noticeably increased just prior to animals reaching the humane endpoint. Treatment of mice with 10 μg of a synthetic 68-amino acid peptide derived from an immunomodulatory molecule secreted by a parasitic worm of humans and livestock, Fasciola hepatica, termed Fasciola hepatica helminth defence molecule (FhHDM), potently suppressed the systemic inflammatory profile, protected mice against acute kidney injury, and improved survival between 48 and 72 h post-procedure. These results suggest that the anti-inflammatory parasite-derived FhHDM peptide has potential as a bio-therapeutic treatment for sepsis.

Surface immobilization and properties optimization of phage hydrolase against Gram-negative bacteria

Immobilized hydrolase not only reduces the production of antibiotic-resistant bacteria, but also effectively improves the stability of hydrolase in external use. In this study, phage hydrolase LysSSE1 against Gram-negative bacteria were surface immobilized and optimized for their bactericidal activity. Different anti-pathogen surface materials were prepared, where LysSSE1 was immobilized on the glass surface with a silica-affinity peptide and into which different peptide linkers were introduced. Immobilized enzymes inserted into the natural amino acid peptide linker exhibited higher bactericidal activity, greater stability, and more consistent bactericidal performance compared to those without the peptide linker. Among these immobilized enzymes, LysSSE1-NL-SiAP1 exhibited the strongest bactericidal activity and the best repeatable bactericidal performance, which only reduced the original performance by about 5% after three bactericidal cycles. Modeling analysis suggested that the presence of peptide linker might increase the molecular flexibility of the proximal hydrolase domain to better interact with the bacterial substrate. Our surface immobilization strategy could be extended to other lytic proteins, providing support for the development of surface sterilization methods and materials.

Profiling Bioactive Components of Natural Eggshell Membrane (NEM) for Cartilage Protection and Its Protective Effect on Oxidative Stress in Human Chondrocytes

The current study aimed to investigate the physicochemical properties of the natural eggshell membrane (NEM) and its protective effects against H2O2-induced oxidative stress in human chondrocytes (SW-1353). Bioactive components from NEM related to cartilage were profiled, consisting of 1.1 ± 0.07% hyaluronic acid, 1.2 ± 0.25% total sulfated glycosaminoglycans as chondroitin sulfate, 3.1 ± 0.33% collagen, and 54.4 ± 2.40% total protein. Protein was hydrolyzed up to 43.72 ± 0.76% using in vitro gastro–intestinal digestive enzymes. Peptides eluted at 9.58, 12.46, and 14.58 min using nano-LC-ESI-MS were identified as TEW, SWVE, and VYL peptides with an M/Z value of 435.1874, 520.2402, and 394.2336, respectively. Radical scavenging activity of NEM at 10 mg/mL using the ABTS assay was revealed to be 2.1 times higher than that of the positive control. NEM treatment significantly enhanced cellular SOD expression (p < 0.05). Pre-treatment with NEM (0.1, 1, and 10 mg/mL) dose-dependently reduced H2O2-induced ROS levels in SW-1353. Cell live imaging confirmed that NEM pre-treatment led to a significant reduction in apoptosis expression compared to control. Results from the present study suggest that NEM rich in cartilage protective components including hyaluronic acid, collagen, and chondroitin antioxidative peptides could be a potential therapeutic agent for osteoarthritis (OA) by scavenging oxidative stress.

Tetraalkynylporphyrin-mediated covalent assembly of gold nanoclusters for targeted tumor fluorescence imaging and enhanced photodynamic therapy.

A covalent assembly strategy was developed to construct agold nanocluster-based nano-assembly (AuNCNA) in a controllable manner, using Au8 nanocluster as node and 5,10,15,20-tetra(4-alkynylphenyl)porphine (TEPP) as ligand. Subsequently, the tripeptide arginine glycine aspartic acid (RGD) peptide is further modified via clicking reaction to build a multi-functional nanoplatform (AuNCNA@RGD) that can integrate the targeted fluorescence imaging and efficient photodynamic therapy (PDT). The strong interregulation of Au8 nanocluster and TEPP results in AuNCNA@RGD exhibiting three distinct advantages: (i) TEPP plays an important role in stabilizing the Au8 nanocluster and keeping the active site fixed within the framework, thereby enhancing stability of Au8 nanocluster; (ii) Au8 nanocluster possess adjustable energy level, which can accelerate the transfer of photogenerated charge and prevent the recombination of electrons and holes, thus improving thephotosensitivity of TEPP for PDT; (iii) AuNCNA exhibits bright fluorescence emission that facilitates RGD-assisted targeted tumor imaging. This work expands the construction method of AuNC assembly, and this assembly method is versatile and can flexibly transform different organic ligands to construct various AuNC-based functional nanomaterials.