Peptide Sequence Research Articles

An Immunoinformatic Approach for Identifying and Designing Conserved Multi-Epitope Vaccines for Coronaviruses

Background/Objectives: The COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has exposed the vulnerabilities and unpreparedness of the global healthcare system in dealing with emerging zoonoses. In the past two decades, coronaviruses (CoV) have been responsible for three major viral outbreaks, and the likelihood of future outbreaks caused by these viruses is high and nearly inevitable. Therefore, effective prophylactic universal vaccines targeting multiple circulating and emerging coronavirus strains are warranted. Methods: This study utilized an immunoinformatic approach to identify evolutionarily conserved CD4+ (HTL) and CD8+ (CTL) T cells, and B-cell epitopes in the coronaviral spike (S) glycoprotein. Results: A total of 132 epitopes were identified, with the majority of them found to be conserved across the bat CoVs, pangolin CoVs, endemic coronaviruses, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Their peptide sequences were then aligned and assembled to identify the overlapping regions. Eventually, two major peptide assemblies were derived based on their promising immune-stimulating properties. Conclusions: In this light, they can serve as lead candidates for universal coronavirus vaccine development, particularly in the search for pan-coronavirus multi-epitope universal vaccines that can confer protection against current and novel coronaviruses.

Proteome Profiling of Cucurbita pepo Phyllosphere After Infection by Podosphaera xanthii and Application of Reynoutria sachalinensis Extract

Podosphaera xanthii is the main causal agent of powdery mildew (PM) disease for Cucurbita pepo. Disease control is attained principally by applications of chemical fungicides, along with parallel use of tolerant crop varieties and alternate application of elicitors to control development of disease resistance. To get insight into C. pepo molecular responses to P. xanthii infection and elicitor treatment we studied the proteomic profile differences at the phyllosphere of a zucchini cultivar susceptible to PM, at the onset of P. xanthii (PX) infection and after application of Reynoutria sachalinensis (RS) plant extract, respectively, using a nano-LC-HRMS/MS, Q-Exactive-Orbitrap approach. Analysis of peptide sequences regarding four treatment groups (Control; PX; RS; and RSPX (PX-infected priorly treated with RS)) resulted in 2070 CuGenDB annotations. Three comparisons (treatments vs. Control) encompassed most of the Differentially Expressed Proteins (DEPs). In these three comparisons, KEGG and Gene Ontology functional analyses highlighted unique differentially enriched pathways—some of which included highly expressed proteins—in PX-related (proteasome, pentose phosphate pathway, and carbon fixation), RS-related (biosynthesis of secondary metabolites, flavonoids, and starch and sucrose metabolism), and RSPX-related (pyruvate metabolism and polycomb repressive complex) comparisons, respectively, suggesting distinct mechanisms of early plant responses modulated by PX and RS. Furthermore, in four out of six comparisons the thiamine metabolism pathway was found to be enriched, suggesting a pivotal role in PX-induced responses.

Antigen-specific T cell frequency and phenotype mirrors disease activity in DRB1*04:04+ rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is associated with high-risk HLA class II alleles known as the "RA shared epitope." Among prevalent shared epitope alleles, study of DRB1*04:04 has been limited. To define relevant epitopes, we identified citrullinated peptide sequences from synovial antigens that were predicted to bind to HLA-DRB1*04:04 and utilized a systematic approach to confirm their binding and assess their recognition by CD4 T cells. After confirming the immunogenicity of 13 peptides derived from aggrecan, cartilage intermediate layer protein (CILP), α-enolase, vimentin, and fibrinogen, we assessed their recognition by T cells from a synovial tissue sample, observing measurable responses to 8 of the 13 peptides. We then implemented a multicolor tetramer panel to evaluate the frequency and phenotype of antigen-specific CD4 T cells in individuals with anti-citrullinated protein antibody (ACPA)-positive RA and controls. In subjects with RA, CILP-specific T cell frequencies were significantly higher than those of other antigens. The surface phenotypes exhibited by antigen-specific T cells were heterogeneous, but Th1-like and Th2-like cells predominated. Stratifying based on disease status and activity, antigen-specific T cells were more frequent and most strongly polarized in RA subjects with high disease activity. In total, these findings identify novel citrullinated epitopes that can be used to interrogate antigen-specific CD4 T cells and show that antigen-specific T cell frequency is elevated in subjects with high disease activity.

Identification and synthesis of a long-chain antimicrobial peptide from the venom of the Liocheles australasiae scorpion.

Scorpion venom contains linear peptides without disulfide bonds in addition to peptides with disulfide bonds. Many such linear peptides have an amphiphilic α-helical structure, often with antimicrobial activity and can be classified into three groups based on their molecular size. Among them, long-chain antimicrobial peptides consisting of more than 40 residues have not been thoroughly studied due to the difficulty of synthesizing them. We have previously reported a transcriptome analysis of the venom gland of Liocheles australasiae that revealed precursor sequences of long-chain antimicrobial peptides. In the study reported here, we identified the mature structure of one such long-chain antimicrobial peptide, LaCT1, which we synthesized using chemical ligation to confirm its structure and evaluate its biological activities. The result showed that LaCT1 exhibited significant antimicrobial activity. In addition, we identified its partial peptides consisting of an N- or C-terminal region, which may be generated by enzymatic cleavage in the venom. Among them, only the peptide containing the N-terminal half region was active. LaCT1 also not only showed insecticidal activity but also synergistically enhanced the effects of another insecticidal peptide identified in L. australasiae venom as well. These results provide insights into the role of antimicrobial peptides in scorpion venom.

Au(I) complexes installed on a self-assembled peptide efficiently catalyze intramolecular cyclization reactions.

Self-assembled peptides are used for diverse applications in the biomedical and technological fields. The morphology and function of the assembled systems are dictated by the peptide sequence and length. In this work, a supramolecular catalyst was obtained upon self-assembly of the diphenylalanine peptide conjugated to a triphenylphosphine Au(I) complex in acetonitrile. The assembled molecules were characterized by spectroscopic techniques and by scanning electron microscopy. The activity of the catalyst was tested on two substrates in cyclization reactions. The morphology and the dimensions of the assembled systems vary depending on the presence of a carboxyl versus an amide C-terminal end. The catalyst efficiently promotes intramolecular cyclization reactions. Results obtained encourage the use of self-assembled peptides for the obtainment of new and efficient catalysts.

Overexpression of TAF4B Promoted the Proliferation of Undifferentiated Spermatogonia in Cattleyak InVitro.

As the hybrid between cattle and yak, cattleyak is a typical male sterile mammal, and the underlying mechanism for its spermatogenic arrest is still unclear. In this study, the coding region of cattleyak TAF4B gene was cloned by RT-PCR and analysed by bioinformatics. To investigate the effects of TAF4B on cellular proliferation and differentiation, an expression vector was generated and introduced into undifferentiated spermatogonia (UDSPG) of cattleyak. The results showed that the protein encoded by TAF4B did not contain the signal peptide sequence. The expression level of TAF4B in UDSPG of cattleyak was lower than that in yak, while the overexpression of TAF4B in cattleyak promoted the proliferation activity of cattleyak UDSPG. Meanwhile, the expression of proliferation and meiosis-related genes was increased but the differentiation-related genes were decreased. Therefore, the aberrant expression of TAF4B in cattleyak UDSPG possibly impaired its proliferation and differentiation equilibrium and decreased its growth potentiality, thereby reducing the quantity of UDSPG and affecting spermatogenesis. This study provided a potential approach for further elucidation of the mechanism of spermatogenesis arrest and provided a new idea for solving the problem of male infertility in cattleyak.

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.

Template‐assisted Flexible‐to‐rigid Transition of Peptides in Head‐to‐tail Self‐polymerization Enables Sequence‐controllable and Post‐modifiable Peptide Nanofibers

AbstractPeptide‐based nanofibers are promising materials for many essential applications and can be generalized into two categories, self‐assembling peptide nanofibers (SAPNs) and poly(amino acid) nanofibers (PAANs). Non‐covalent SAPNs are sequence‐controllable, but poorly stable and not suitable for post‐modification. While covalent PAANs are post‐modifiable, however, their sequences are either monotonic or undefined. The nanofibers obtained by head‐to‐tail covalent coupling polymerization of sequence‐known peptides, which we call series‐connected peptide nanofibers (SCPNs), promise to have the advantages of both SAPNs and PAANs, but they are barely reported. The undesired backbiting effect during the head‐to‐tail polymerization is one of the possible challenges. Here, we present a template‐assisted strategy to trigger the flexible‐to‐rigid transition of peptide units, which can avoid the backbiting effect and enable consecutive intermolecular polymerization of peptides to produce desired sequence‐controlled covalent SCPNs. SCPNs are highly stable and can function as excellent parent materials for various post‐processing to create diverse hierarchical materials independent of the peptide sequence. Moreover, SCPNs allow for the display of predetermined functional groups at regular intervals along the nanofibers by pre‐modification of the initial peptide sequence. SCPNs represent a new category of peptide‐based nanofibers with outstanding performances and vast potential.

Distinct Effects of SARS-CoV-2 Protein Segments on Structural Stability, Amyloidogenic Potential, and α-Synuclein Aggregation.

Amyloidosis is characterized by the abnormal accumulation of misfolded proteins, called amyloid fibrils, leading to diverse clinical manifestations. Recent studies on the amyloidogenesis of SARS-CoV‑2 protein segments have raised concerns on their potential link to post-infection neurodegeneration, however, the mechanisms remain unclear. Herein, we investigated the structure, stability, and amyloidogenic propensity of a nine-residue segment (SK9) of the SARS-CoV-2 envelope protein and their impact on neuronal protein α-synuclein (αSyn) aggregation. Specifically, the amino acid sequence of the SK9 wildtype has been modified from a basic and positively charged peptide (SFYVYSRVK), to a nearly neutral and more hydrophobic peptide (SAAVASAVK, labelled as SK9 var1), and to an acidic and positively charged peptide (SFYVYSRVK, labelled as SK9 var2). Our findings reveal that the SK9 wildtype exhibited a pronounced amyloidogenic propensity due to its disordered and unstable nature, while the SK9 variants possessed more ordered and stable structures preventing the amyloid formation. Significantly, the SK9 wildtype demonstrated distinct effect on αSyn aggregation kinetics and aggregate morphology to facilitate the formation of αSyn aggregates with enhanced resistance against enzymatic degradation. This study highlights the potential of modifying short peptide sequences to fine-tune their properties, providing insights into understanding and regulating viral-induced amyloid aggregations.

Discovery of Lacto-N-Biosidases and a Novel N-Acetyllactosaminidase Activity in the CAZy Family GH20: Functional Diversity and Structural Insights.

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.

Enhances the resistance of rice to lepidopteran pests by fusing the Cry1Ca and Cry2Aa genes with self-cleavage peptide sequence.

Accumulation of two or more Bacillus thuringiensis (Bt) proteins in plant not only improves the resistance to pests and broadens the resistance spectrum of crops, but also delays the development of pest resistance. The self-cleavage peptide sequence was used to link two codon-optimized genes, so as to achieve simultaneous accumulation of two low homologous insecticidal proteins in one plant. The rice transformants accumulating Cry1Ca and Cry2Aa proteins were fed to local lepidopteran pests and the larva mortality in 5 days were 100%. The sum of Cry1Ca and Cry2Aa proteins in leaves of transformants E1C&2A-1 and E2A&1C-18 were 10.60 and 9.55 μg g-1 fresh weight (FW), respectively, and the larva mortality of fall armyworm fed on their leaves for 5 days reached 100%. For the control transformants that expressed one Bt protein, the content of Cry1Ca in leaves of transformant E1CM031 was 14.94 μg g-1 FW, and that of Cry2Aa in leaves of transformant B2A4008S was 11.90 μg g-1 FW, but the larva mortality of fall armyworm fed on leaves of E1CM031 and B2A4008S for 5 days were 77.78% and 52.78%, respectively. Although the total Bt contents in transformants expressing one Bt protein were higher than that of transformants expressing two Bt proteins, the lethality of transformants expressing one Bt protein were obviously lower than that of transformants expressing two Bt proteins. The lethal effect of accumulating both Cry1Ca and Cry2Aa proteins in rice was stronger than that of amassing Cry1Ca or Cry2Aa protein only, which meant there was synergistic effect between Cry1Ca and Cry2Aa proteins. © 2024 Society of Chemical Industry.

Modulation of biomineralization morphology by phosphorylated collagen peptides: insights into osteogenesis imperfecta pathophysiology.

Osteogenesis imperfecta (OI) is a hereditary skeletal disorder characterized by bone fragility and deformities, primarily attributed to defects in type I collagen, the most abundant structural protein in humans. Multiple phosphorylation sites have been detected within collagen, suggesting that phosphorylation may influence mineralization processes, thereby impacting the development of OI. In this study, we investigated the modulation of biomineralization morphology by phosphorylated collagen peptides mimicking Gly-Ser mutations in osteogenesis imperfecta. A series of collagen peptide sequences, including GPO13S, GPO13pS, GPO12S, GPO12pS, GPO11S, and GPO11pS, were synthesized to explore the role of phosphorylation in peptide stability and its templating effect on biomineralization. The CD results indicated that the phosphorylation of Gly-pSer mutants reduces the stability of collagen peptides. SEM images revealed that phosphorylated peptides acted as templates, guiding the morphology of calcium carbonate into either olive-like or spherical structures, depending on their conformational state of the peptides. Non-phosphorylated peptides maintained a calcite crystal structure. The XRD patterns predominantly exhibited peaks associated with calcite and vaterite for GPO13pS-CaCO3, GPO12pS-CaCO3, and GPO11pS-CaCO3, and peaks associated with calcite for GPO13S-CaCO3, GPO12S-CaCO3, and GPO11S-CaCO3, indicating a transformation of mesocrystals influenced by peptide phosphorylation. Our findings elucidate the crucial role of phosphorylated collagen peptides in mediating biomineralization morphology and polymorph selection, offering insights into the complex pathophysiology of OI.

Discovery of Highly Bioactive Peptides through Hierarchical Structural Information and Molecular Dynamics Simulations.

Peptide drugs play an essential role in modern therapeutics, but the computational design of these molecules is hindered by several challenges. Traditional methods like molecular docking and molecular dynamics (MD) simulation, as well as recent deep learning approaches, often face limitations related to computational resource demands, complex binding affinity assessments, extensive data requirements, and poor model interpretability. Here, we introduce PepHiRe, an innovative methodology that utilizes the hierarchical structural information in peptide sequences and employs a novel strategy called Ladderpath, rooted in algorithmic information theory, to rapidly generate and enhance the efficiency and clarity of novel peptide design. We applied PepHiRe to develop BH3-like peptide inhibitors targeting myeloid cell leukemia-1, a protein associated with various cancers. By analyzing just eight known bioactive BH3 peptide sequences, PepHiRe effectively derived a hierarchy of subsequences used to create new BH3-like peptides. These peptides underwent screening through MD simulations, leading to the selection of five candidates for synthesis and subsequent in vitro testing. Experimental results demonstrated that these five peptides possess high inhibitory activity, with IC50 values ranging from 28.13 ± 7.93 to 167.42 ± 22.15 nM. Our study explores a white-box model driven technique and a structured screening pipeline for identifying and generating novel peptides with potential bioactivity.

A Universal Support for the Solid-Phase Synthesis of Peptidyl-tRNA Mimics.

Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are often required for structural and functional studies of protein synthesis at the ribosome. These conjugates can be synthesized by solid-phase chemical synthesis, which allows maximum flexibility in both the peptide and RNA sequence. The commonly used strategy is based on (3'-N-aminoacyl)-3'-amino-3'-deoxyadenosine solid supports, which already contain the first C-terminal amino acid of the target peptidyl chain. This is a limitation in the sense that different individual supports must be synthesized for different C-terminal amino acids. In this study, we demonstrate a solution to this problem by introducing a novel universal support. The key is a free ribose 3'-NH2 group that can be coupled to any amino acid. This is made possible by a photocleavable ether moiety that links the ribose 2'-O to the support, thus avoiding the typical O-to-N migration that occurs when using 2'-O-acyl linked solid supports. Once assembled, the conjugate is readily cleaved by UV irradiation. The structural integrity of the obtained peptidyl-RNA conjugates was verified by mass spectrometry analysis. In conclusion, the new photocleavable solid support makes the synthesis of 3'-peptidyl tRNA mimics of different peptidyl chains significantly more efficient compared to the commonly used approaches.

In-silico and in-vitro study of novel antimicrobial peptide AM1 from Aegle marmelos against drug-resistant Staphylococcus aureus

Antimicrobial peptides have garnered increasing attention as potential alternatives due to their broad-spectrum antimicrobial activity and low propensity for developing resistance. This is for the first time; proteome sequences of Aegle marmelos were subjected to in-silico digestion and AMP prediction were performed using DBAASP server. After screening the peptides on the basis of different physiochemical property, peptide sequence GKEAATKAIKEWGQPKSKITH (AM1) shows the maximum binding affinity with − 10.2 Kcal/mol in comparison with the standard drug (Trimethoprim) with − 7.4 kcal/mol and − 6.8 Kcal/mol for DHFR and SaTrmK enzyme respectively. Molecular dynamics simulation performed for 300ns, it has been found that peptide was able to stabilize the protein more effectively, analysed by RMSD, RMSF, and other statistical analysis. Free binding energy for DHFR and SaTrmK interaction from MMPBSA analysis with peptide was found to be -47.69 and − 44.32 Kcal/mol and for Trimethoprim to be -13.85 Kcal/mol and − 11.67 Kcal/mol respectively. Further in-vitro study was performed against Methicillin Susceptible Staphylococcus aureus (MSSA), Methicillin Resistant Staphylococcus aureus (MRSA), Multi-Drug Resistant Staphylococcus aureus (MDR-SA) strain, where MIC values found to be 2, 4, and 8.5 µg/ml lesser in comparison to trimethoprim which has higher MIC values 2.5, 5, and 9.5 µg/ml respectively. Thus, our study provides the insight for the further in-vivo study of the peptides against multi-drug resistant S. aureus.

Antiviral Peptide-Generative Pre-Trained Transformer (AVP-GPT): A Deep Learning-Powered Model for Antiviral Peptide Design with High-Throughput Discovery and Exceptional Potency

Traditional antiviral peptide (AVP) discovery is a time-consuming and expensive process. This study introduces AVP-GPT, a novel deep learning method utilizing transformer-based language models and multimodal architectures specifically designed for AVP design. AVP-GPT demonstrated exceptional efficiency, generating 10,000 unique peptides and identifying potential AVPs within two days on a GPU system. Pre-trained on a respiratory syncytial virus (RSV) dataset, AVP-GPT successfully adapted to influenza A virus (INFVA) and other respiratory viruses. Compared to state-of-the-art models like LSTM and SVM, AVP-GPT achieved significantly lower perplexity (2.09 vs. 16.13) and higher AUC (0.90 vs. 0.82), indicating superior peptide sequence prediction and AVP classification. AVP-GPT generated a diverse set of peptides with excellent novelty and identified candidates with remarkably higher antiviral success rates than conventional design methods. Notably, AVP-GPT generated novel peptides against RSV and INFVA with exceptional potency, including four peptides exhibiting EC50 values around 0.02 uM—the strongest anti-RSV activity reported to date. These findings highlight AVP-GPT’s potential to revolutionize AVP discovery and development, accelerating the creation of novel antiviral drugs. Future studies could explore the application of AVP-GPT to other viral targets and investigate alternative AVP design strategies.

Photochemical and Collision-Induced Cross-Linking in Stereochemically Distinct Scaffolds of Peptides and Nitrile Imines in Gas-Phase Ions.

Intramolecular cross-linking between peptides and nitrile-imine intermediates was studied in stereochemically distinct conjugates in which the reacting components were mounted on cis-1,2-cyclohexane and trans-1,4-cyclohexane scaffolds that we call 1,2-s-peptides and 1,4-s-peptides, respectively. The nitrile-imine intermediates were generated by N2 loss from 2,5-diaryltetrazole tags upon UV-photodissociation at 213 and 250 nm or by collision-induced dissociation, and further interrogated by CID and UVPD-MS3. Peptide fragment ion series originating from linear structures and macrocyclic cross-links were distinguished and used to quantify the cross-linking yields. The yields in MS2 varied between 27% for AAAG conjugates to 78% for GAAAK conjugates, depending on the peptide sequence. The CID-MS3 yields were in a 57-97% range, depending on the peptide sequence. Structures of 1,2-s-peptide and 1,4-s-peptide ions as well as several of their nitrile-imine intermediates and cross-links were investigated by high-resolution cyclic ion mobility in combination with Born-Oppenheimer molecular dynamics and density functional theory calculations. Matches between the experimental and calculated collision cross sections and ion relative Gibbs energies were used to assign peptide structures. Peptide conjugates C-terminated with Gly and Lys residues underwent cross-linking by the carboxyl group, as established by MS3 sequencing and corroborated by carboxyl blocking experiments that lowered the cross-linking yields. Peptide conjugates C-terminated with Arg also cross-linked via the side-chain guanidine group. A notable feature of the 1,4-s-peptide ions was the participation of low-energy twist-boat cyclohexane conformers that was enforced by strong hydrogen bonds between the peptide and nitrile imine.

Advanced Hydrogels: Enhancing Tissue Bioengineering with RGD Peptides and Carbon Nanomaterials.

The advancement of tissue engineering (TE) is driven by the development of scaffolds that mimic the mechanical, spatial, and biological environment of the extracellular matrix (ECM), crucial for regulating cell behavior and tissue repair. Hydrogels, 3D networks of polymer chains, are particularly suited for TE due to their high biocompatibility, ability to mimic tissue water content, facilitate cell migration, sustain growth factor release, and offer controllable physical properties. However, hydrogels mimicking the ECM often face challenges related to cell adhesion due to the absence of specific receptors. This issue can be addressed by incorporating ECM components into the polymer matrix, such as the peptide sequence arginine-glycine-aspartic acid (RGD), known for its role in cell adhesion. Additionally, carbon nanomaterials (CNMs) offer unique physicochemical properties that can improve scaffold-cell interactions. Despite the potential benefits, there are limited reports on their combination. RGD-CNM hydrogels enable a more accurate emulation of the natural cellular environment, enhancing tissue engineering applications. This hybrid approach may promote robust cell adhesion along with exceptional mechanical and electrical properties. This review outlines the potential benefits of these hybrid scaffolds and their synergistic potential, aiming to inspire new research directions in this innovative field.

Efficiency Enhancement in Peptide Hydrogelators: The Crucial Role of Side Chain Hydrogen Bonding Over Aromatic π-π Interactions.

Short peptide assemblies that form supramolecular hydrogels are stabilized by both intermolecular noncovalent interactions among amino acid side chains and hydrogen bonding between peptide backbone amides. Previous research has emphasized the inclusion of aromatic amino acids in short peptide sequences, positing that aromatic π-π interactions contribute significantly to inducing efficient hydrogelation i.e., at low minimum gelation concentrations (MGCs). However, herein, we demonstrate that additional hydrogen bonding interactions from amino acid side chains play a more pivotal role in the efficiency of peptide hydrogelation compared to aromatic π-π interactions. We investigated two sets of Fluorenylmethoxycarbonyl (Fmoc)-functionalized α-synuclein and human islet amyloid peptide fragments [Fmoc-NVGGAVVT (Syn-N) and Fmoc-NFGAIL (IAP-N)], substituting asparagine (N) with phenylalanine (Syn-F/IAP-F), alanine (Syn-A/IAP-A), and glutamine (Syn-Q/IAP-Q). This allowed us to explore the effects of aromatic (π-system), aliphatic (hydrophobic), and hydrogen bonding effects with varying chain lengths on hydrogel formation. Our results reveal that Syn-N and Syn-Q exhibit MGC of 0.03 and 0.05 wt %, respectively, classifying them as super hydrogelators (MGC < 0.1 wt %). These values are 4.0-6.6-fold lower than Syn-F and Syn-A, with Syn-N demonstrating greater efficiency than Syn-Q. Similarly, IAP-N exhibited a substantial decrease in MGC by 8.75, 3.75, and 2.5 folds compared to IAP-A, IAP-F, and IAP-Q, respectively. Experimental evidence and molecular dynamic simulation suggest that -CO-NH2 of asparagine side chains effectively engaged in hydrogen bonding, thereby immobilizing water molecules at low gelator concentrations. Although glutamine shares similar -CO-NH2 functionality, its hydrogelation efficiency is less pronounced compared to asparagine, likely due to its longer alkyl chain, which may hinder the formation of a hydrogen bonding network in the self-assembled structure compared to asparagine-containing peptides. These findings offer valuable insights for designing efficient peptide hydrogelators or lowering MGCs by substituting amino acids with asparagine/glutamine in peptide sequences. Additionally, modifying peptide properties through asparagine/glutamine substitution could optimize hydrogel properties for specific applications.

Tailored 3D Agarose-Well Integrated with Human Skin Equivalents for Enhanced Skin Penetration Assessment

We developed a tailored 3D Agarose-well system integrated with reconstructed human skin equivalents to enhance skin penetration assessments. This system addresses common limitations in traditional trans-well reconstructions, such as dermal layer contraction and limited lateral diffusion, by entangling collagen fibrils within the Agarose-well. We evaluated the penetration behavior of three peptides, with and without skin-penetrating peptide (SPP) sequences, alongside adenosine, a known anti-wrinkle agent. Despite a SPP having a molecular weight approximately four times greater than that of adenosine, its kinetic constant was similar, with values of about 39 and 34, respectively. Moreover, this living skin equivalent system not only allowed for the evaluation of adenosine penetration, but also demonstrated its biological effects, with adenosine significantly enhancing procollagen synthesis by approximately 23% compared to the control. Overall, this novel strategy holds the potential for tailoring 3D Agarose-wells and advancing high-performance gel development, making it a promising approach for applications in tissue engineering, medical science, regenerative medicine, and cosmetics.