Synthetic Peptides Research Articles

Activation of polycystin-1 signaling by binding of stalk-derived peptide agonists

Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Detection and Localization of IL-8 and CXCR1 in Rainbow Trout Larvae in Response to Pseudomonas aeruginosa Lipopolysaccharide

The salmonid industry faces challenges due to the susceptibility of fish to opportunistic pathogens, particularly in early developmental stages. Understanding the immunological capacity during these stages is crucial for developing effective disease control strategies. IL-8R, a member of the G-protein-coupled receptor family, acts as a receptor for Interleukin 8 (IL-8). The binding of IL-8 to IL-8R plays a major role in the pathophysiology of a wide spectrum of inflammatory conditions. This study focused on the immune response capacity of rainbow trout (Oncorhynchus mykiss) larvae by analyzing IL-8/CXCR1 response to lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Previous research demonstrated that LPS from P. aeruginosa acts as a potent immunostimulant in teleost, enhancing pro-inflammatory cytokines. The methodology included in silico analysis and the synthesis and characterization of an omCXCR1-derived epitope peptide, which was used to produce omCXCR1-specific anti98 serum in mice. The research revealed that rainbow trout larvae 19 days post-hatching (dph) exhibited pronounced immune responses post-stimulation with 1 µg/mL of LPS. This was evidenced by the upregulated protein expression of IL-8 and omCXCR1 in trout larvae 2 and 8 h after LPS challenge, as analyzed by ELISA and immunohistochemistry. Furthermore, fluorescence microscopy successfully revealed the colocalization of IL-8 and its receptor in cells from mucosal tissues after LPS challenge in larvae 19 dph. These findings underscore the efficacy of LPS immersion as a method to activate the innate immune system in trout larvae. Furthermore, we propose IL-8 and its receptor as molecular markers for evaluating immunostimulation in the early developmental stages of salmonids.

Sec-isoamyl Mercaptan (SIT), a Multi-faceted Disulfide Based Protecting Group for Cysteine Thiol

IntroductionThe successful synthesis of a peptide requires the synchronization of several processes, including the efficient execution of protecting group chemistry. For cysteine (Cys)-peptides, this is more crucial because the trifunctional Cys has a free thiol in its side chain. During synthesis, this free thiol function remains protected with suitable protecting groups and can be removed after synthesis using appropriate methods. Sec-isoamyl mercaptan (SIT) is a versatile disulfide-based protecting group for Cys side chain thiol. The removal of SIT from Cys thiol can be achieved using a mild reducing agent (e. g. DTT). This later promotes efficient disulfide bond formation by oxidation. SIT can also direct/activate the Cys thiol for the chemoselective formation of disulfide bonds by thiol-disulfide interchange.MethodsPeptides were synthesized using solid-phase peptide synthesis techniques. The removal of the SIT group was carried out either in the solid phase or in the solution.ResultsIn the present study, we have shown that SIT can be efficiently removed both in solution and on-resin to facilitate disulfide-bridged peptide synthesis. This was exemplified by two syntheses of an atosiban derivative, where the SIT was removed in solution or in solid-phase. Furthermore, a SIT-based facile one-pot synthesis pathway was devised for disulfide-rich peptides. The strategy was faster and greener as it did not involve using an oxidizer. Conotoxin (two S–S) and linaclotide amide (three S–S) were successfully synthesized by adopting the SIT-based strategy. Finally, a racemization study was carried out for SIT, Trt and StBu-protected Cys-peptides. In all cases, SIT-protected peptides showed lesser racemization than StBu-protected peptides. In some instances (synthesis using DMF), SIT-protected peptides showed less racemization compared to the Trt congeners.ConclusionOverall, the multifaceted use of SIT-protection during the synthesis of disulfide-rich peptides has illustrated its versatility as a Cys thiol protecting group.

Innovative On-Resin and in Solution Peptidomimetics Synthesis via Metal-Free Photocatalytic Approach.

Nowadays, peptidomimetics are widely studied, being useful tools in drug discovery and medicinal chemistry. The coupling between a carboxylic acid with an amine to form a peptide bond is the most common reaction to obtain peptides/peptidomimetics. In this work, we have investigated an innovative metal-free photoredox-catalyzed carbamoylation to form peptidomimetics thanks to the reaction between dihydropyridines functionalized with amino acids (or peptide sequences) and differently functionalized imines. As the organic photocatalyst, we used 4CzIPN, a donor-acceptor cyanoarene vastly used in photoredox catalysis. By easily modulating the amino acid (or peptide sequence), which is directly attached to the dihydropyridine, we proposed this key-reaction as new valuable method to obtain peptidomimetics, in situ building the not-natural portion of the sequence. Moreover, we successfully employed this methodology in solid phase peptide synthesis, both inserting the new photoredox-generated amino acid at the end or in the middle of the sequence. Peptides with different lengths and secondary structures were prepared, proving the success of this approach, even in sterically hindered environment. Herein, to the best of our knowledge, we describe the first photocatalytic protocol which allows the building of the peptide backbone, with the possibility of simultaneously inserting a non-coded amino acid in the sequence.

Adaptation to space conditions of novel bacterial species isolated from the International Space Station revealed by functional gene annotations and comparative genome analysis

BackgroundThe extreme environment of the International Space Station (ISS) puts selective pressure on microorganisms unintentionally introduced during its 20+ years of service as a low-orbit science platform and human habitat. Such pressure leads to the development of new features not found in the Earth-bound relatives, which enable them to adapt to unfavorable conditions.ResultsIn this study, we generated the functional annotation of the genomes of five newly identified species of Gram-positive bacteria, four of which are non-spore-forming and one spore-forming, all isolated from the ISS. Using a deep-learning based tool—deepFRI—we were able to functionally annotate close to 100% of protein-coding genes in all studied species, overcoming other annotation tools. Our comparative genomic analysis highlights common characteristics across all five species and specific genetic traits that appear unique to these ISS microorganisms. Proteome analysis mirrored these genomic patterns, revealing similar traits. The collective annotations suggest adaptations to life in space, including the management of hypoosmotic stress related to microgravity via mechanosensitive channel proteins, increased DNA repair activity to counteract heightened radiation exposure, and the presence of mobile genetic elements enhancing metabolism. In addition, our findings suggest the evolution of certain genetic traits indicative of potential pathogenic capabilities, such as small molecule and peptide synthesis and ATP-dependent transporters. These traits, exclusive to the ISS microorganisms, further substantiate previous reports explaining why microbes exposed to space conditions demonstrate enhanced antibiotic resistance and pathogenicity.ConclusionOur findings indicate that the microorganisms isolated from ISS we studied have adapted to life in space. Evidence such as mechanosensitive channel proteins, increased DNA repair activity, as well as metallopeptidases and novel S-layer oxidoreductases suggest a convergent adaptation among these diverse microorganisms, potentially complementing one another within the context of the microbiome. The common genes that facilitate adaptation to the ISS environment may enable bioproduction of essential biomolecules need during future space missions, or serve as potential drug targets, if these microorganisms pose health risks.17AcJHQ5oWAqHhb9n7GQHSVideo

Complementary Strategies for Installation of Thioimidates into Peptide Backbones.

Thioimidates are a precursor and synthetic branch point to access either thioamide or amidine isosteres of the native amide (peptide bond). Previous syntheses of thioimidate-containing peptides were prone to side reactivity and required slow, cumbersome steps that were difficult to monitor. We describe a more efficient approach to directly couple thioimidates onto the growing peptide chain. This work also outlines optimal conditions for thioimidate formation on solid support and identifies potential off-target sites for alkylation that impact the choice of protecting group.

Application of Sortase-Mediated Ligation for the Synthesis of Block Copolymers and Protein-Polymer Conjugates.

Sortase-mediated ligation (SML) has become a powerful tool for site-specific protein modification. However, sortaseA (SrtA) suffers from low catalytic efficiency and mediates an equilibrium reaction. Therefore, ligations with large macromolecules may be challenging. Here, the synthesis of polymeric building blocks for sortase-mediated ligation constituting peptide-polymers with either the recognition sequence for sortaseA (LPX1TGX2) or its nucleophilic counterpart (Gx) is demonstrated. The peptide-polymers are synthesized by solid-phase peptide synthesis followed by photo-iniferter (PI) reversible addition-fragmentation chain-transfer (RAFT) polymerization of various monomers. The building blocks are subsequently utilized to investigate possibilities and limitations when using macromolecules in SML. In particular, diblock copolymers are obtained even when using the orthogonal building blocks in equimolar ratio by exploiting a technique to shift the reaction equilibrium. However, ligations of two polymers can not be achieved when the degree of polymerization exceeds 100. Subsequently, C-terminal protein-polymer conjugates are synthesized. Several polymers are utilized that can replace the omnipresent polyethylene glycol (PEG) in future therapeutics. The conjugation is exemplified with a nanobody that is known for efficient neutralization of SARS-CoV-2. The study demonstrates a universal approach to polymer-LPX1TGX2 and Gx-polymer building blocks and gives insight into their application in SML.

A-144 Liquid chromatography multiple reaction monitoring-based assay for quantitative assessment of fibrinogen levels in clinical samples

Abstract Background Fibrinogen plays a critical role in hemostasis and is essential to clot formation. Changes in the quantity or activity of fibrinogen can lead to disorders affecting coagulation. Thus, it is essential to precisely quantify fibrinogen levels to differentiate between hyperfibrinogenemia in hypercoagulable disorders and dysfibrinogenemia, hypo/afibrinogenemia, and irregular bleeding. Current diagnostic tests that use antibodies to detect fibrinogen or activity-based measures are susceptible to interference from heparin contamination, hemolysis, and hyperbilirubinemia. Here, we evaluated the use of targeted proteomics for developing a quantitative assay for fibrinogen in plasma samples. Methods Four peptides each for alpha and gamma chains of fibrinogen were synthesized using FMOC chemistry on a Liberty Blue (CEM Corp) peptide synthesizer. A targeted multiple reaction monitoring (MRM) assay was developed and optimized using Dionex Ultimate 3000 HPLC connected to a TSQ Altis triple quadrupole mass spectrometer (Thermo Scientific). Evaluations were conducted using a range of standards, such as pure protein, WHO plasma and commercially available plasma with known fibrinogen amounts for the accurate quantitation of fibrinogen. Calibration curves were generated using different dilutions in various matrices. The repeatability and precision of the assay were evaluated across five different days. The plasma samples were processed and digested in a 96-well plate and subjected to C18 clean-up using a Bravo automated liquid handler (Agilent). All samples were spiked-in with corresponding heavy-labeled peptides prior to digestion with trypsin. Multiple reaction monitoring assays were performed on tryptic digests of plasma samples. Results Calibration curves using commercial plasma for all peptides showed linearity from 20 mg/dl to 1,308 mg/dl. This covers the entire clinical range including normal (200-350 mg/dl), deficient states (<100 mg/dl) and excess production (>350 mg/dl) that can be used for evaluation of bleeding disorders including consumption coagulopathy and hyperfibrinogenemia. Based on the overall performance in terms of linearity, limit of detection/quantitation, carryover and coefficient of variation (CV) across five different days, two peptides per chain were finally selected for developing the quantitative assay. The intra-assay variability was <5% and intra-day CV was ∼20%. This assay is currently being tested on a variety of clinical samples representing a range of fibrinogen levels. Conclusions Our study focused on assessing whether multiplexed MRM assay could be implemented to measure fibrinogen levels in plasma. Our data indicate the feasibility of MRM-based assays for determining fibrinogen levels as an alternative to existing non-mass spectrometry-based activity or antigen assays.

Synthesis and characterization of new antimicrobial peptides derived from Temporin F.

Antimicrobial peptides (AMPs) are a promising source of new compounds against resistant bacteria. Temporins are a class of AMPs found on the amphibian Rana temporaria and show activity against Gram-positive and Gram-negative bacteria. There are few studies on how these antimicrobials have been used, but new Temporin-F derivatives were engineered with Lys-substitutions to assess the impact of the net charge on antimicrobial activity and toxicity. We demonstrated through some assays that it is possible to increase the antibacterial activity while maintaining a reduced peptide hemolytic activity with specific substitutions. Our lead synthetic peptide, G6K-Temporin F, has shown higher antimicrobial activity against Gram-negative and Gram-positive bacteria in vitro (MIC range 2 to 32 μmol L-1), with low hemolytic activity maintained, resulting in an increase in the therapeutic window (TW), of 12.5. Also, it showed more resistant to enzymatic degradation. On the other hand, more significant increases in net charges, such as in P3K-G11K-Temporin F, result in a severe increase in toxicity with lower gains in antimicrobial activity (TW of 0.65). In conclusion, we demonstrated that a moderate increase in net charge can lead to a more active analog and G6K-Temporin F is revealed to be promising as a candidate for new AMP therapeutics.

Translation of Deoxyribonucleic Acid into Synthetic Alpha Helical Peptides for Darwinian Evolution.

DNA-encoded libraries connect the phenotypes of synthetic molecules to a DNA barcode; however, most libraries do not tap into the potential of Darwinian evolution. Herein, we report a DNA-templated synthesis (DTS) architecture to make peptides that are stabilized into α-helical conformations via head-to-tail supramolecular cyclization. Using a pilot library targeting MDM2, we show that repeated screening can amplify a binder from the lowest abundance in the library to a ranking that correlates to binding affinity. The study also highlights the need to design libraries such that the chemistry avoids biases from the heterogeneous yield in DTS.

A cofactor-induced repressive type of transcription factor condensation can be induced by synthetic peptides to suppress tumorigenesis.

Transcriptional factors (TFs) act as key determinants of cell death and survival by differentially modulating gene expression. Here, we identified many TFs, including TEAD4, that form condensates in stressed cells. In contrast to YAP-induced transcription-activating condensates of TEAD4, we found that co-factors such as VGLL4 and RFXANK alternatively induced repressive TEAD4 condensates to trigger cell death upon glucose starvation. Focusing on VGLL4, we demonstrated that heterotypic interactions between TEAD4 and VGLL4 favor the oligomerization and assembly of large TEAD4 condensates with a nonclassical inhibitory function, i.e., causing DNA/chromatin to be aggregated and entangled, which eventually impede gene expression. Based on these findings, we engineered a peptide derived from the TEAD4-binding motif of VGLL4 to selectively induce TEAD4 repressive condensation. This "glue" peptide displayed a strong antitumor effect in genetic and xenograft mouse models of gastric cancer via inhibition of TEAD4-related gene transcription. This new type of repressive TF phase separation exemplifies how cofactors can orchestrate opposite functions of a given TF, and offers potential new antitumor strategies via artificial induction of repressive condensation.

The In vitro evaluation of in silico-designed synthetic peptides AKVUAM-1 and AKVUAM-2 on human lung fibroblast cell line MRC5 and Mycobacterium tuberculosis isolates

Tuberculosis is a major global health problem caused by Mycobacterium tuberculosis and the increase in drug resistance is driving the need for new treatments. Today, various approaches are being applied in the development of drugs for the treatment of tuberculosis. Computer-aided drug design (CADD) enables the prediction of pharmacological efficacy for potential drug molecules during the design process. Thus, new therapeutic compounds can be developed that are more potent, less toxic and have fewer side effects than existing drugs. In this study, we investigated the in vitro activities of AKVUAM-1 and AKVUAM-2 synthetic peptides designed in silico by computer-aided drug design method to inhibit the interaction between M. tuberculosis outer membrane protein Cpn T and macrophage surface receptor CR-1 and Surfactant D protein. Notably, these synthetic peptides do not show cytotoxic effect on normal lung tissue and do not kill M. tuberculosis directly. The MIC values for AKVUAM-1 were higher than 512 μg/ml for all bacterial strains except IST-16 strain (128 μg/ml). According to our results, AKVUAM-1 and AKVUAM-2 synthetic peptides have the potential to be successful candidates for investigating their potential to block macrophage entry of bacilli as targeted.

Serum antibodies against mimotopes of Merkel cell polyomavirus oncoproteins detected by a novel immunoassay in healthy individuals and Merkel cell carcinoma patients.

Merkel cell polyomavirus (MCPyV) is the foremost causative factor of Merkel cell carcinoma (MCC), a rare yet highly aggressive skin cancer. Although the evaluation of circulating IgG antibodies against Merkel cell polyomavirus (MCPyV) LT/sT oncoproteins is clinically useful for MCC diagnosis/prognosis, a limited number of assays for identifying such antibodies have been developed. Herein, a novel indirect immunoassay with synthetic epitopes/mimotopes of MCPyV oncoproteins was computationally designed and experimentally validated on control sera and sera from healthy individuals and MCC patients. Upon computational design of five synthetic peptides, the performance of the immunoassay in detecting anti-oncoprotein IgGs in MCPyV-positive and -negative control sera was evaluated. The immunoassay was afterwards extended on sera from healthy individuals, and, for longitudinal analysis, MCC patients. Performance properties such as sensitivity and specificity and positive/negative predictive values were adequate. Receiver-operating characteristic (ROC) curves indicated that the areas under the curves (AUCs) were within the low/moderately accurate ranges. Immunoassay was repeatable, reproducible and accurate. As expected, the serum anti-oncoprotein IgG prevalence in healthy individuals was low (2%-5%). Anti-oncoprotein IgGs slightly increased when MCC patients experienced partial tumour remission and/or stable disease, compared to baseline. Our data indicate that the newly developed immunoassay is reliable for detecting circulating anti-oncoprotein IgGs both in healthy individuals and MCC patients.

Amide and peptide synthesis via nickel-catalyzed cross-electrophile coupling

Amide and peptide synthesis via nickel-catalyzed cross-electrophile coupling

Insight into Protein Engineering: From In silico Modelling to In vitro Synthesis.

Protein engineering alters the polypeptide chain to obtain a novel protein with improved functional properties. This field constantly evolves with advanced in silico tools and techniques to design novel proteins and peptides. Rational incorporating mutations, unnatural amino acids, and post-translational modifications increases the applications of engineered proteins and peptides. It aids in developing drugs with maximum efficacy and minimum side effects. Currently, the engineering of peptides is gaining attention due to their high stability, binding specificity, less immunogenic, and reduced toxicity properties. Engineered peptides are potent candidates for drug development due to their high specificity and low cost of production compared with other biologics, including proteins and antibodies. Therefore, understanding the current perception of designing and engineering peptides with the help of currently available in silico tools is crucial. This review extensively studies various in silico tools available for protein engineering in the prospect of designing peptides as therapeutics, followed by in vitro aspects. Moreover, a discussion on the chemical synthesis and purification of peptides, a case study, and challenges are also incorporated.

Synthesis and Evaluation of amyloid beta peptide/ Ruthenium III -based complex drugs as drug delivery and anticancer activity

The development and characterization of anticancer complex drugs (ACD), specifically Amyloid Beta Peptide (ABP) - Ruthenium III (Ru III) - nivolumab (NB), were explored through analytical techniques. Fourier-transform infrared (FTIR) spectroscopy demonstrated the structural transformation of peptides from α-helical to β-sheet formations, aligning with amyloid fibril aggregation. Ruthenium (III) complex synthesis was confirmed through distinct absorption peaks in FTIR analysis. High-resolution scanning electron microscopy (HRSEM) revealed the fibrous and smooth morphology of ACD, while thermogravimetric analysis (TGA) confirmed the decomposition stages and stability of the ruthenium complexes. The encapsulation efficiency and in vitro release profile of nivolumab (NB) within ABP-RuIII-NB were investigated, showing a two-phase release over 40h. Cytotoxicity studies using acridine orange and ethidium bromide staining techniques indicated significant apoptosis in human oral squamous cell carcinoma (OSCC) -treated cells. These findings highlight the potential of ABP-RuIII-NB as an effective cancer treatment with controlled drug release and high cytotoxicity against cancer cells.

Exploring novel antioxidant cyclic peptides in corn protein hydrolysate: Preparation, identification and molecular docking analysis

Antioxidant cyclic peptides were successfully identified from a corn protein hydrolysate. Hydrolysate by Alcalase + Flavourzyme showed the highest cyclic peptide purity (48.36 ± 1.81 %) and higher antioxidant activities compared with other hydrolysate. The success of peptide cyclization in hydrolysate was demonstrated by thermogravimetric analysis and thin-layer chromatography (TLC) analysis. Thermogravimetric analysis showed that the thermal stability of hydrolysate after cyclization was significantly increased, which was related to the formation of cyclic peptides. Peptides with molecular weight less than 1000 Da accounted for more than 80 % in hydrolysate after cyclization. After separation using gel silica chromatography and semi-preparative reverse phase high performance liquid chromatography (RP-HPLC), 22 novel antioxidant cyclic peptides were identified by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) and orbitrap-tandem mass spectrometry (Orbitrap-MS/MS). Synthetic cyclic peptides with the same sequence were synthesized and characterized for their antioxidant activity. Molecular docking suggested that the free radical molecules could bind with the cyclic backbone and side chain of cyclic peptides through hydrogen bonding, hydrophobic interaction as well as electrostatic interaction. This study has important implications for the high-value utilization of corn protein and new cyclic peptides drugs or functional food development.

Functional characterization of a nanobody-based glycoprotein VI-specific platelet agonist

BackgroundGlycoprotein (GP)VI is a platelet-specific collagen receptor required for platelet activation during hemostasis. Platelet reactivity toward collagen is routinely assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with suspensions of fibrillar collagen or synthetic cross-linked collagen-related peptide (CRP-XL). However, these suspensions are poorly standardized or difficult to produce. Nanobodies are small recombinant camelid-derived heavy-chain antibody variable regions. They are highly stable, specific, and ideal candidates for developing a stable GPVI agonist for diagnostic assays. ObjectivesDevelop a stable nanobody-based GPVI agonist. MethodsNanobody D2 (NbD2) was produced as dimers and purified. Tetramers were generated via C-terminal fusion of dimers with click chemistry. Nanobody constructs were functionally characterized with light transmission aggregometry (LTA) in platelet-rich plasma and whole blood flow cytometry. Diagnostic performance was assessed in patients with inherited platelet function disorders with LTA and flow cytometry. ResultsNbD2 was specific for human platelet GPVI. Dimers did not result in platelet activation in LTA or flow cytometry settings and fully inhibited CRP-XL-induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen-induced platelet aggregation in platelet-rich plasma. However, NbD2 tetramers caused full platelet aggregation, as well as P-selectin expression and fibrinogen binding. NbD2 tetramers were able to discriminate between inherited platelet function disorder patients and healthy controls based on fibrinogen binding, similar to CRP-XL. ConclusionNanobody tetramers to GPVI induce platelet activation and can be used to assess the GPVI pathway in diagnostic assays.

Enhanced Osteogenesis in 2D and 3D Culture Systems Using RGD Peptide and α-TCP Phase Transition within Alginate-Based Hydrogel.

Cell-laden hydrogels have been extensively investigated in various tissue engineering fields bytheir potential capacity to deposit numerous types of cells in a specific area. They are largely used in soft-tissue engineering applications because of their low mechanical strength. In addition, sodium alginate is well-known for its encapsulation, loading capacity and for being easily controllable; however, it lacks cell-binding ligands and hence the ability to adhere cells. In this study, it is aimed to enhance osteogenesis in cells encapsulated in alginate and improve its mechanical properties by introducing a synthetic peptide and calcium phosphate phase transition. To increase cell-hydrogel interactions and increasing cell viability, an RGD peptide is added to a photocrosslinkable methacrylate-modified alginate, and alpha-tricalcium phosphate (α-TCP) is added to the hydrogel to increase its mechanical strength via phase transition. Cell proliferation, growth, and differentiation are assessed in both 2D and 3D cell cultures. The addition of α-TCP significantly improved the mechanical properties of the hydrogel. Moreover, the RGD peptide and α-TCP showed a synergistic effect with significantly improved cell adhesion and osteogenesis in both 2D and 3D cell cultures. Therefore, the functional hydrogel developed in this study can potentially be used for bone tissue regeneration.

GSP1-111 Modulates the Microglial M1/M2 Phenotype by Inhibition of Toll-like Receptor 2: A Potential Therapeutic Strategy for Depression.

Neuroinflammation plays a vital role in neurodegenerative diseases and neuropsychiatric disorders, and microglia and astrocytes chiefly modulate inflammatory responses in the central nervous system (CNS). Toll-like receptors (TLRs), which are expressed in neurons, astrocytes, and microglia in the CNS, are critical for innate immune responses; microglial TLRs can regulate the activity of these cells, inducing protective or harmful effects on the surrounding cells, including neurons. Therefore, regulating TLRs in microglia may be a potential therapeutic strategy for neurological disorders. We examined the protective effects of GSP1-111, a novel synthetic peptide for inhibiting TLR signaling, on neuroinflammation and depression-like behavior. GSP1-111 decreased TLR2 expression and remarkably reduced the mRNA expression of inflammatory M1-phenotype markers, including tumor necrosis factor (TNF)α, interleukin (IL)-1β, and IL-6, while elevating that of the M2 phenotype markers, Arg-1 and IL-10. In vivo, GSP1-111 administration significantly decreased the depression-like behavior induced by lipopolysaccharide (LPS) in a forced swim test and significantly reduced the brain levels of M1-specific inflammatory cytokines (TNFα, IL-1β, and IL-6). GSP1-111 prevented the LPS-induced microglial activation and TLR2 expression in the brain. Accordingly, GSP1-111 prevented inflammatory responses and induced microglial switching of the inflammatory M1 phenotype to the protective M2 phenotype. Thus, GSP1-111 could prevent depression-like behavior by inhibiting TLR2. Taken together, our results suggest that the TLR2 pathway is a promising therapeutic target for depression, and GSP1-111 could be a novel therapeutic candidate for various neurological disorders.