Peptide Block Research Articles

Papain-Mediated Conjugation of Peptide Nucleic Acids to Delivery Peptides: A Density Functional Theory/Molecular Mechanics Metadynamics Study in Aqueous and Organic Solvent.

Enzymatic peptide synthesis is a powerful alternative to solid-phase methods, as enzymes can have high regio- and stereoselectivity and high yield and require mild reaction conditions. This is beneficial in formulation research due to the rise of nucleic acid therapies. Peptide nucleic acids (PNAs) have a high affinity toward DNA and RNA, and their solubility and cellular delivery can be improved via conjugation to peptides. Here, we designed and assessed the viability of the papain enzyme to conjugate four PNA-peptide models in water and an organic solvent using QM/MM metadynamics. We found that the reactions in water yield better results, where three conjugates could potentially be synthesized by the enzyme, with the first transition state as the rate-limiting step, with an associated energy of 14.53 kcal mol-1, although with a slight endergonic profile. The results highlight the importance of considering the enzyme pockets and different substrate acceptivities and contribute to developing greener, direct, and precise synthetic routes for nucleic acid-based therapies. By exploring the enzyme's potential in conjunction with chemical synthesis, current protocols can be simplified for the synthesis of longer nucleic acids and peptide sequences (and, by extension, proteins) from smaller oligo or peptide blocks.

Selector of amino-acid scales set.

Experimental and theoretical properties of amino acids as building blocks of peptides and proteins have been extensively researched. Each such method assigns a number to each amino acid, and one such assignment is called amino-acid scale. Their usage in bioinformatics to explain and predict behaviour of peptides and proteins is of essential value. The number of such scales is very large. There are more than a hundred scales related just to hydrophobicity. A large number of scales can be a computational burden for algorithms that try to define peptide descriptors combining several of these scales. Hence, it is of interest to construct a smaller, but still representative set of scales. Here, we present software that does this. We test it on the set of scales using a database constructed by Kawashima and collaborators and show that it is possible to significantly reduce the number of scales observed without losing much of the information. An algorithm is implemented in C#. As a result, we provide a smaller database that might be a very useful tool for the analyses and construction of new peptides. Another interesting application of this database would be to compare the artificial intelligence construction of peptides having as an input the complete Kawashima database and this reduced one. Obtaining in both cases similar results would give much credibility to the constructs of such AI algorithms.

C-Terminal Decarboxylation of Proline-Derived Building Blocks for Protein-Binding Peptides.

Using a set of conformationally restricted Proline-derived Modules (ProMs), our group has recently succeeded in developing inhibitors for the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain, which is a key mediator of cell migration and plays an important role in tumor metastasis. While these (formally) pentapeptidic compounds show nanomolecular binding affinities towards EVH1, their drug-like properties and cell permeability need to be further optimized before they can be clinically tested as therapeutic agents against metastasis. In this study, we sought to improve these properties by removing the C-terminal carboxylic acid function of our peptoids, either by late-stage decarboxylation or by direct synthesis. For late-stage decarboxylation of ProM-like systems, a method for reductive halo decarboxylation was optimized and applied to several proline-derived substrates. In this way, a series of new decarboxy ProMs suitable as building blocks for decarboxy EVH1 inhibitors were obtained. In addition, we incorporated decarboxy-ProM-1 into the pentapeptide-like compound Ac[2ClF][ProM-2][Decarb-ProM-1], which showed similar affinity towards EVH1 as the methyl ester derivative (Ac[2Cl-F][ProM-2][ProM1]OMe). However, despite better calculated drug-like properties, this compound did not inhibit chemotaxis in a cellular assay.

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered “Crab”-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. “Crab”-like configuration of two catalytic peptides (“pincers”) flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for “single-pincer” conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled “Crabs” to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5′-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

Milk exosomes anchored with hydrophilic and zwitterionic motifs enhance mucus permeability for applications in oral gene delivery.

Exosomes have emerged as a promising tool for the delivery of drugs and genetic materials, owing to their biocompatibility and non-immunogenic nature. However, challenges persist in achieving successful oral delivery due to their susceptibility to degradation in the harsh gastrointestinal (GI) environment and impeded transport across the mucus-epithelium barrier. To overcome these challenges, we have developed high-purity bovine milk exosomes (mExo) as a scalable and efficient oral drug delivery system, which can be customized by incorporating hydrophilic and zwitterionic motifs on their surface. In our study, we observed significantly improved transport rates by 2.5-4.5-fold in native porcine intestinal mucus after the introduction of hydrophilic and zwitterionic surface modifications, as demonstrated by transwell setup and fluorescence recovery after photobleaching (FRAP) analysis. Remarkably, mExo functionalized by a block peptide (BP), consisting of cationic and anionic amino acids arranged in blocks at the two ends, demonstrated superior tolerability in the acidic gastric environment (with a protein recovery rate of 84.8 ± 7.7%) and exhibited a 2.5-fold increase in uptake by intestinal epithelial cells. Furthermore, both mExo and mExo-BP demonstrated successful intracellular delivery of functional siRNA, resulting in up to 65% suppression of the target green fluorescence protein (GFP) gene expression at a low dose of siRNA (5 pmol) without causing significant toxicity. These findings highlight the immense potential of modifying mExo with hydrophilic and zwitterionic motifs for effective oral delivery of siRNA therapies.

Engineering tunable dual peptide hybrid coatings promote osseointegration of implants

Utilizing complementary bioactive peptides is a promising surface engineering strategy for bone regeneration on osteogenesis. In this study, we designed block peptides, (Lysine)6-capped RGD (K6-(linker-RGD)3) and OGP (K6-linker-(YGFGG)2), which were mildly grafted onto PC/Fe-MPNs through supramolecular interactions between K6 and PC residues on the MPNs surface to form a dual peptide coating, named PC/Fe@K6-RGD/OGP. The properties of the block peptides coating, including mechanics, hydrophilicity, chemical composition, etc., were detailly characterized by various techniques (ellipsometry, quartz crystal microbalance, X-ray photoelectron spectroscopy, water contact angle, scanning electronic microscopy and atomic force microscopy). Importantly, the RGD/OGP ratio can be well adjusted, which allowed optimizing the RGD/OGP ratio to endow significantly enhanced osteogenic activity of MC3T3-E1 cells through the Wnt/β-catenin pathway, while also promoting cell adhesion, immune regulation, inhibiting osteoclast differentiation and oxidative stress reduction. In vivo, the optimized RGD/OGP coatings promoted bone regeneration and osseointegration around implants in rats with bone defects. In conclusion, rationally designed PC/Fe@K6-RGD/OGP coating integrated RGD and OGP bioactivities, providing a convenient approach to enhance bioinert implant surfaces for bone regeneration.

A Transformable Supramolecular Bispecific Cell Engager for Augmenting Natural Killer and T Cell-Based Cancer Immunotherapy.

Immune cells are pivotal in cancer immunotherapy, yet their therapeutic effectiveness is often hampered by limited tumor infiltration and inhibitory tumor microenvironments. An alkaline phosphatase (ALP)-responsive and transformable supramolecular bis-specific cell engager (Supra-BiCE) to harness natural killer (NK)/T cells for effective cancer immunotherapy is introduced here. The Supra-BiCE, consisting of both SA-P (a phosphorylated peptide targeting and blocking programmed cell death ligand 1 (PD-L1)) and SA-T (a phosphorylated peptide targeting and blocking T cell immunoglobulin and ITIM domain (TIGIT)) is constructed by a simple co-assembling strategy. Upon intravenous administration, Supra-BiCE self-assembles into nanoribbons and interacts with NK/T cells via TIGIT. Notably, these nanoribbons undergo transformation into long nanofibrils within ALP-overexpressing tumor regions, resulting in enhanced binding affinities of Supra-BiCE to both PD-L1 and TIGIT. Consequently, this leads to the accumulation and retention of NK/T cells within tumor regions. Furthermore, the combinatorial blockade of checkpoints by Supra-BiCE activates infiltrating NK/T cells. Moreover, the adjustable peptide ratio in Supra-BiCE enables customization for optimal therapeutic effects against distinct tumor types. Particularly, Supra-BiCE (T:P = 1:3) achieved 98.27% tumor suppression rate against colon carcinoma model. Overall, this study offers a promising tool for engaging NK and T cells for cancer immunotherapy.

Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses.

Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive. We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses. Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs. These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Epitope identification of a Lys63 linkage ubiquitin antibody by mass spectrometric epitope excision and extraction approaches.

Ubiquitin, a conserved protein in eukaryotic cells, exists as a monomer or polyubiquitin chains known as isopeptide-linked polymers. These chains are attached to a substrate or other ubiquitin molecules through a covalent bond between the α-amino group of lysine in ubiquitin and glycine in the C-terminal of the subsequent ubiquitin unit. The choice of the specific lysine residue in ubiquitin for forming ubiquitin-ubiquitin chains determines its biochemical and biological function. A detailed chemical structure-function evaluation of the respective polyubiquitin chain is required. Interestingly, specific lysine linkage polyubiquitin chains become covalently bonded to many pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so the interaction between polyubiquitin chains and ubiquitin antibodies is very useful. For example, the neurofibrillary tangles of Alzheimer's disease and the Lewy bodies seen in Parkinson's disease are heavily ubiquitinated and can be readily visualized using specific ubiquitin antibodies. This study utilized synthetic ubiquitin building block peptides that contained various lysine residues (K6, K11, K33, K48, and K63) linked to a Gly-Gly dipeptide, with the aim of exploring the recognition specificity of the Lys63-polyubiquitin antibody. The interaction studies between different ubiquitin building blocks and the specific Lys63-ubiquitin (K63-Ub) antibody were performed by affinity-mass spectrometry (Affinity-MS) and immunoblotting which enables direct protein identification from biological material with unprecedented selectivity. Affinity-MS and dot blot data proved the specific binding of the K63-Ub antibody to the ubiquitin peptides containing Lys6 or Lys63 residues. In epitope excision for mass spectrometric epitope identification, the ubiquitin building block with Lys63 residue bound to the immobilized K63-Ub antibody was proteolytically cleaved using pronase. The resulting epitope and non-epitope fractions were subjected to matrix-assisted laser desorption/ionization-time of flight analysis, revealing that the epitope is located within the sequence ubiquitin(60-66). Epitope extraction-MS consistently confirmed these findings.

Creation of Artificial Luciferase 60s from Sequential Insights and Their Applications to Bioassays.

In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors' previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.

Effect of Charged Block Length Mismatch on Double Diblock Polyelectrolyte Complex Micelle Cores.

Polyelectrolyte complex micelles are hydrophilic nanoparticles that self-assemble in aqueous environments due to associative microphase separation between oppositely charged blocky polyelectrolytes. In this work, we employ a suite of physical characterization tools to examine the effect of charged block length mismatch on the equilibrium structure of double diblock polyelectrolyte complex micelles (D-PCMs) by mixing a diverse library of peptide and synthetic charged-neutral block polyelectrolytes with a wide range of charged block lengths (25-200 units) and chemistries. Early work on D-PCMs suggested that this class of micelles can only be formed from blocky polyelectrolytes with identical charged block lengths, a phenomenon referred to as chain length recognition. Here, we use salt annealing to create PCMs at equilibrium, which shows that chain length recognition, a longstanding hurdle to repeatable self-assembly from mismatched polyelectrolytes, can be overcome. Interestingly, D-PCM structure-property relationships display a range of values that vary systematically with the charged block lengths and chemical identity of constituent polyelectrolyte pairings and cannot be described by generalizable scaling laws. We discuss the interdependent growth behavior of the radius, ionic pair aggregation number, and density in the micelle core for three chemically distinct diblock pairings and suggest a potential physical mechanism that leads to this unique behavior. By comparing the results of these D-PCMs to the scaling laws recently developed for single diblock polyelectrolyte complex micelles (S-PCMs: diblock + homopolymer), we observe that D-PCM design schemes reduce the size and aggregation number and restrict their growth to a function of charged block length relative to S-PCMs. Understanding these favorable attributes enables more predictive use of a wider array of charged molecular building blocks to anticipate and control macroscopic properties of micelles spanning countless storage and delivery applications.

IGF1c mimetic peptide-based supramolecular hydrogel microspheres synergize with neural stem cells to promote functional recovery from spinal cord injury

Spinal cord injuries (SCI) often cause severe inflammatory response and neuronal loss, which consequently deteriorates motor function. Although exogenous stem cells are considered to be vital seed cells for cell replacement therapy after SCI, their low survival rate and low efficiency of neuronal differentiation after in vivo transplantation limit their widespread application in clinical practice. To this end, this study proposed a new strategy for dramatically improving the efficiency of insulin-like growth factor 1 (IGF1c) mimetic peptide to activate its receptor by fabricating IGF1c mimetic peptide supramolecular hydrogel microspheres (IGF1c-PHM) using an innovatively designed piezoelectric ceramic-driven thermal electrospray device. IGF1c-PHM displayed an average size of 10 µm and could activate the IGF1R of neural stem cells (NSCs) more effectively than IGF1c peptide block hydrogel (IGF1c-PH). In vitro, IGF1c-PHM exerted strong neuroprotective effects by maintaining the proliferation, inhibiting the apoptosis of NSCs, and promoting the efficiency of differentiation into neurons in presence of myelin extracts. In vivo, IGF1c-PHM improved the inflammatory environment by suppressing the recruitment of the inflammatory cells, altering the subtypes of the macrophages, and promoting the survival rate of endogenous cells and NSCs in the injury area. In particular, IGF1c-PHM enhanced the proliferation ability and neuronal differentiation of NSCs in vivo after SCI. In the presence of IGF1c-PHM, the transplanted NSCs could differentiate into several mature neurons at the injury site, and the axons regenerated in the injury site were remyelinated, all of which closely correlate with the improvement of the electrophysiological indices after SCI. Finally, at 8 weeks post-SCI, the gait analysis and behavioral scores confirmed the significant improvement in motor function in rats treated with IGF1c-PHM and NSCs. Therefore, IGF1c-PHM combined with NSCs is a promising therapy for treating SCI.

A Tandem‐Locked Fluorescent NETosis Reporter for the Prognosis Assessment of Cancer Immunotherapy

AbstractNETosis, the peculiar type of neutrophil death, plays important roles in pro‐tumorigenic functions and inhibits cancer immunotherapy. Non‐invasive real‐time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a Tandem‐locked NETosis Reporter 1 (TNR1) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker‐specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem‐locked design allows TNR1 to differentiate NETosis from neutrophil activation, while single‐locked reporters fail to do so. The near‐infrared signals from activated TNR1 in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near‐infrared signals from activated TNR1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor‐bearing living mice, but also proposes a generic approach for tandem‐locked probe design.

A Tandem-Locked Fluorescent NETosis Reporter for the Prognosis Assessment of Cancer Immunotherapy.

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer immunotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a Tandem-locked NETosis Reporter 1 (TNR1 ) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR1 to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR1 in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design.

In Situ STING-Activating Nanovaccination with TIGIT Blockade for Enhanced Immunotherapy of Anti-PD-1-Resistant Tumors.

Immunotherapies comprising programmed cell death protein 1/PD ligand 1 (PD-1/PD-L1) immune checkpoint inhibitors are effective cancer treatments. However, the low response rate and immunoresistance resulting from alternative immune checkpoint upregulation and inefficient immune stimulation by T cells are problematic. The present report describes a biomimetic nanoplatform that simultaneously blocks the alternative T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) checkpoint and activates the stimulator of interferon genes (STING) signaling pathway in situ for enhanced antitumor immunity. The nanoplatform is engineered by fusing a red blood cell membrane with glutathione-responsive liposome-encapsulated cascade-activating chemoagents (β-lapachone and tirapazamine), and anchoring them with a detachable TIGIT block peptide (named as RTLT). In the tumor environment, the peptide is spatiotemporally released to reverse T-cell exhaustion and restore antitumor immunity. The cascade activation of chemotherapeutic agents causes DNA damage and inhibits the repair of double-stranded DNA, which induces robust in situ STING activation for an efficient immune response. The RTLT inhibits anti-PD-1-resistant tumor growth, and prevents tumor metastasis and recurrence in vivo by inducing antigen-specific immune memory. This biomimetic nanoplatform thus provides a promising strategy for in situ cancer vaccination.

Therapeutic targeting of CNBP phase separation inhibits ribosome biogenesis and neuroblastoma progression via modulating SWI/SNF complex activity

Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive. Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used. Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients. These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.

Flexibility, size and hydrophobicity of alkyl side groups in methoxy-poly (ethylene glycol)-polypeptide for the nano-assembly and thermo-sensitivity

Tailoring of amino acid residues is a feasible strategy to design the structure of peptide for special application. We selected poly(ethylene glycol) and α-amino acids with hydrophobic alkyl side group (L-alanine，L-valine and L-leucine) to synthesize di-block co-polypeptide. The copolymers with methyl side group self-assembled into random coil in majority and aggregated further into stable spherical nanoarchitectures with a little filament owing to the small size, less flexibility and less hydrophobicity of methyl group. By lengthening peptide block, the copolymer self-organized into overwhelming β-sheet and ordered nanorods or nanofibers due to the strengthening hydrophobicity as the copolymers with isopropyl side group did. The copolymers with isobutyl side groups showed cloudy solutions and irregular morphology because the flexibility and large size of isobutyl groups hampered the regular stacking of β-plated sheets and agglomerated into micro-sized precipitates despite of the most hydrophobicity. As temperature rising, both the transition of random coil to β-sheet and PEG dehydration contributed to the sol-gel transition for the copolymers with methyl side group, while PEG dehydration was the primary reason of sol-gel transition for the copolymers with isopropyl side group. In this article, we first disclosed the self-organization process of mPEG-b-polypeptide, which would lay the foundation for the designing of polypeptide-related materials with well-defined properties.

Tissue inhibitor of metalloproteinase (TIMP)-1 predicts negative remodeling and recovery of ejection fraction in acute heart failure with reduced ejection fraction

Abstract Background Heart failure with recovered ejection fraction (HFrecEF) is a recently identified phenotype of heart failure, which had lower rates of mortality, heart failure hospitalization, ventricular assist device placement, and heart transplantation compared to heart failure with reduced ejection fraction (HFrEF). Several predictors of HFrecEF had been proposed such as female gender, lower HF duration, nonischemic cause, no left bundle branch block (LBBB), lower EF, and lower N-terminal pro-brain natriuretic peptide (NT-proBNP) in the previous studies. Tissue inhibitors of metalloproteinase-1 (TIMP-1) was a biomarker that regulated the activity of metalloproteinases (MMPs). TIMP-1 was up-regulated during HF and was associated with ventricular remodeling and poor prognosis. However, the predictive value of TIMP-1 and MMP-9 in recovery of EF was yet to be investigated. Purpose The present study aimed at investigating the predictive value of TIMP-1 and MMP-9 of recovery of EF. Methods Subjects with acute decompensated heart failure and reduced left ventricular ejection fraction were eligible for this study. HFrEF was defined as EF <40% and HFrecEF was defined as follow-up EF ≥40% and ≥10% improvement from baseline EF. The biomarkers including NT-proBNP, plasma MMP-9 and serum TIMP-1 were measured in all enrolled subjects at admission. The receiver operating characteristic (ROC) curves and Youden index were used to select the model variables and cutoff values. Binary logistic regression analysis assessed the association between the biomarkers and recovery ejection fraction. Results Among a total of 91 AHF with reduced ejection fraction subjects (70.1±16.2 years, baseline EF 28.9±7.6%), 19 (20.8%) HFrecEF and 72 (79.2%) persistent HFrEF were found at 6 months. TIMP-1 and NT-proBNP were significantly higher in persistent HFrEF compared to HFrecEF. MMP-9 was similar between these two phenotypes of HF. The area under the ROC curve for recovery of ejection fraction of TIMP-1, MMP-9 and NT-proBNP were 0.69, 0.52, and 0.65, respectively. The optimal cutoff value of TIMP-1 according to Youden index was 200.68 ng/ml. In binary logistic regression analysis, TIMP-1 was negatively correlated with recovered ejection fraction as continuous variables [per 1-SD=8.96 ng/ml, odds ratio and 95% confidence interval: 0.99 (0.98–1.00)] and categorical variables [cutoff value 200.68 ng/ml, odds ratio and 95% confidence interval: 0.16 (0.05–0.54)] after adjusting age, gender, EF, acute myocardial infarction, LBBB and NT-proBNP. Conclusions In AHF subjects with HFrEF, TIMP-1 was negatively correlated to the recovery of EF and could be a potential biomarker for the prediction of the recovery of EF. Funding Acknowledgement Type of funding sources: None.

Human Claudin-Derived Peptides Block the Membrane Fusion Process of Zika Virus and Are Broad Flavivirus Inhibitors.

ABSTRACTZika virus (ZIKV) is a mosquito-borne flavivirus that emerged in the Pacific islands in 2007 and spread to the Americas in 2015. The infection remains asymptomatic in most cases but can be associated with severe neurological disorders. Despite massive efforts, no specific drug or vaccine against ZIKV infection is available to date. Claudins are tight-junction proteins that favor the entry of several flaviviruses, including ZIKV. In this study, we identified two peptides derived from the N-terminal sequences of claudin-7 and claudin-1, named CL7.1 and CL1.1, respectively, that inhibited ZIKV infection in a panel of human cell lines. Using cell-to-cell fusion assays, we demonstrated that these peptides blocked the ZIKV E-mediated membrane fusion. A comparison of the antiviral efficacy of CL1.1 and CL7.1 pointed to the importance of the peptide amphipathicity. Electron microscopic analysis revealed that CL1.1 altered the ultrastructure of the viral particles likely by binding the virus lipid envelope. However, amphipathicity could not fully explain the antiviral activity of CL1.1. In silico docking simulations suggested that CL1.1 may also interact with the E protein, near its stem region. Overall, our data suggested that claudin-derived peptides inhibition may be linked to simultaneous interaction with the E protein and the viral lipid envelope. Finally, we found that CL1.1 also blocked infection by yellow fever and Japanese encephalitis viruses but not by HIV-1 or SARS-CoV-2. Our results provide a basis for the future development of therapeutics against a wide range of endemic and emerging flaviviruses.IMPORTANCE Zika virus (ZIKV) is a flavivirus transmitted by mosquito bites that have spread to the Pacific Islands and the Americas over the past decade. The infection remains asymptomatic in most cases but can cause severe neurological disorders. ZIKV is a major public health threat in areas of endemicity, and there is currently no specific antiviral drug or vaccine available. We identified two antiviral peptides deriving from the N-terminal sequences of claudin-7 and claudin-1 with the latter being the most effective. These peptides block the envelope-mediated membrane fusion. Our data suggested that the inhibition was likely achieved by simultaneously interacting with the viral lipid envelope and the E protein. The peptides also inhibited other flaviviruses. These results could provide the basis for the development of therapies that might target a wide array of flaviviruses from current epidemics and possibly future emergences.

Identification of the Binding Epitope of an Anti-mouse CCR4 Monoclonal Antibody, C4Mab-1.

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.