P53 Peptide Research Articles

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200–350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251–305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262–279. Amino acid 262–279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262–273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

Efficacy of the Vaccine Candidate Based on the P0 Peptide against Dermacentor nitens and Ixodes ricinus Ticks.

The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine.

Towards a High-Affinity Peptidomimetic Targeting Proliferating Cell Nuclear Antigen from Aspergillus fumigatus.

Invasive fungal infections (IFIs) are prevalent in immunocompromised patients. Due to alarming levels of increasing resistance in clinical settings, new drugs targeting the major fungal pathogen Aspergillus fumigatus are required. Attractive drug targets are those involved in essential processes like DNA replication, such as proliferating cell nuclear antigens (PCNAs). PCNA has been previously studied in cancer research and presents a viable target for antifungals. Human PCNA interacts with the p21 protein, outcompeting binding proteins to halt DNA replication. The affinity of p21 for hPCNA has been shown to outcompete other associating proteins, presenting an attractive scaffold for peptidomimetic design. p21 has no A. fumigatus homolog to our knowledge, yet our group has previously demonstrated that human p21 can interact with A. fumigatus PCNA (afumPCNA). This suggests that a p21-based inhibitor could be designed to outcompete the native binding partners of afumPCNA to inhibit fungal growth. Here, we present an investigation of extensive structure-activity relationships between designed p21-based peptides and afumPCNA and the first crystal structure of a p21 peptide bound to afumPCNA, demonstrating that the A. fumigatus replication model uses a PIP-box sequence as the method for binding to afumPCNA. These results inform the new optimized secondary structure design of a potential peptidomimetic inhibitor of afumPCNA.

Structural and Functional Cardiac Parameters in Patients with AL Amyloidosis Correlate with Uptake of the Amyloidophilic Radiotracer Iodine ( 124I) Evuzamitide

Background: The deposition of fibrillar aggregates of monoclonal free light chains in the heart is a characteristic feature of light chain associated (AL) amyloidosis. Cardiac amyloid is the major cause of morbidity and mortality in these patients. Structural and functional sequelae of cardiac amyloidosis can be appreciated clinically using echocardiography, cardiac magnetic resonance imaging, and measurement of serum NT-proBNP and troponins. These measures are not particularly amyloid-specific, and the correlation between parameters derived from these evaluations and amyloid burden remain enigmatic. The iodine-124 labeled peptide (p5+14), iodine ( 124I) evuzamitide, is a novel radiotracer that is being evaluated for the detection of systemic amyloidosis, of any type, by PET/CT imaging. As an amyloid binding peptide, this radiotracer has the potential to serve as a first line diagnostic agent capable of providing whole body semi-quantitative estimation of organ-specific amyloid load. Nascent studies of the relationship between 124I-evuzamitide uptake and established clinical parameters in patients with amyloidosis are underway. Here we report a single site, open label pilot study of 18 ( n=18) patients with AL ( n=8) and ATTR amyloidosis ( n=10) that evaluates the cardiac uptake of 124I-evuzamitide with contemporaneous echocardiographic and serum biomarkers (NCT05968846). Methods: The data described herein was collected as part of an open label single site repeat imaging study that plans to enroll up to 20 ( n=20) patients with AL ( n=10) and ATTR ( n=10) amyloidosis. To date, eight ( n=8) patients with AL and ten ( n=10) with ATTR amyloidosis have undergone 124I-evuzamitide PET/CT imaging with contemporaneous transthoracic echocardiography and assessment of serum NTproBNP within ~30 h of imaging. Patients received 37 or 74 MBq 124I-evuzamitide, and PET/CT imaging was performed at ~5 h post injection. Images were interpreted by a single reviewer, experienced in evaluating this tracer, for positive cardiac uptake. A fully manual 2D region of interest (ROI) analysis was performed encompassing the free left ventricular (LV) wall, LV lumen and interventricular septum (IVS). Blood pool was used as a reference tissue to determine standard uptake value ratios (SUVR mean) and to control for the rate of tracer clearance from the central compartment. Cardiac uptake of radiotracer was compared with free LV wall thickness (cm), IVS thickness (cm), global longitudinal strain (GLS, %), and serum NT-proBNP (pg/mL) measurements. Pearson and Spearman correlations were used to test for associations between variables. Results: Patients with AL amyloidosis were 2012 Mayo Stage I ( n=2/8) or Stage II ( n=6/8). The mean serum NTproBNP was 672.9±762.4 pg/mL (min=98, max=2327). With singular exception, all patients with AL amyloidosis had visually evident cardiac uptake of 124I-evuzamitide in the PET/CT images. In patients with AL amyloidosis ( n=8), the SUVR mean (encompassing the LV, lumen and IVS) correlated strongly with LV thickness (cm) (Pearson r=0.83, p=0.011) and GLS (%) (Pearson r=0.74, p=0.037), but not the IVS thickness (cm) (Pearson r=0.57, p=0.142). In contrast, in patients with ATTR ( n=10), there was no significant correlation of SUVR mean with GLS ( p=0.395), but a moderate significant correlation of SUVR mean with the LV ( p=0.037) and IVS ( p=0.017) thickness was observed. Similarly, in this pilot cohort, we observed a strong positive correlation between serum NTproBNP and cardiac SUVR mean (Pearson r=0.81, p=0.015), but not for patients with ATTR amyloidosis (Pearson r=0.58, p=0.079). Conclusion: Serum NTproBNP is a validated and qualified measure of overall survival for patients with AL amyloidosis (PMID: 27416985). Global longitudinal strain, with or without inclusion of other structural parameters such as left atrial volume, is a strong predictor of survival in patients with AL amyloidosis (PMID: 29111123, PMID: 37143609). PET/CT imaging using 124I-evuzamitide is a promising technique for diagnosing and monitoring AL and other types of amyloidosis. In light of these early results that demonstrate a strong relationship between validated metrics of all cause mortality in patients with AL, larger studies should seek to evaluate similar relationships with some metric of 124I-evuzamitide uptake in the heart, and indeed other organs.

TFP5, a Peptide Derived from Cdk5 Activator p35, Protects Pancreatic β Cells from Glucose Toxicity.

We studied the effect of TFP5 on MIN6 cells (cultured mouse islet β cells) treated with different concentrations of glucose (5 or 25 mM). The results were verified in C57BL/6J mice (control; n=12) and db/db mice with type 2 diabetes mellitus (n=12). To synthesize TFP5, peptide p5 (a derivative of p35 protein, activator of cyclin-dependent kinase 5, Cdk5) was conjugated with a FITC tag at the N-terminus and an 11-amino acid TAT protein transduction domain at the C-terminus. TFP5 was employed to inhibit Cdk5 activity and then to evaluate its efficiency in treating experimental type 2 diabetes mellitus. TFP5 effectively inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected pancreatic β cells from apoptosis in vitro and in vivo. In addition, TFP5 inhibited inflammation in pancreatic islets by reducing the expression of inflammatory cytokines TGF-β1, TNFα, and IL-1β. These novel data indicates that TFP5 is a promising candidate for treatment of type 2 diabetes mellitus.

Design and In-silico Screening of Short Antimicrobial Peptides (AMPs) as Anti-Tubercular Agents Targeting INHA

Introduction: Tuberculosis has been studied as a potentially serious bacterial infection affecting the lung caused by Mycobacterium tuberculosis. In addition to its severe impact on health, resistance to existing drugs has also been seen as a rising concern in the space of medicinal solutions. Therapeutic peptides have the potential to complement existing drug designs to provide effective outcomes against Mycobacterium tuberculosis-resistant strains. Methods: This study illustrated a computational approach to design and test peptides against NADHdependent enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis. A human antimicrobial peptide LL-37 was used as a template, and a further 6 peptides were designed, and their binding and interactions against NADH-dependent enoyl-acyl carrier protein reductase were examined. Further, toxicity, immunogenicity, and a broad spectrum of physicochemical properties were calculated to evaluate the therapeutic and safety profile of these peptides. Results: These peptides were structurally modelled and docked with the protein to determine their binding poses and affinity. The molecular interaction of LL-37 with protein was treated as a reference to evaluate the effectiveness of designed peptides. Solvent accessible surface area (SASA) and ΔG binding free energy of docked complexes assisted in the ranking of these peptides. Eventually, peptides P1: LLGDFFRKSKEK, P3: LLFGDRFLLKEK and P7: LLGDFFRLLKEK were selected for 100 ns molecular dynamic simulation as they showed predicted dissociation constants of 8.7×10-4 M, 3.3×10-4 M and 1.2×10-4 M, respectively. These peptides showed direct hydrogen bond formation with ILE21 and LYS165, which are critical active site residues of the protein. The structural variation pattern collected from the MD simulation suggested a strong and stable binding of P3 and P1 with the protein with RMSD 4-5 Å with the starting conformation under the non-fluctuating state. These two peptides showed relatively similar binding results compared with the control peptide LL-37. Comprehensive structural analysis was performed for the middle structures of the most populated cluster generated from 100 ns MD simulation trajectory. Conclusion: Later, MMPBSA binding energies of these structures were computed, where the average binding free energies of P1, P3, and P7 peptides were -146.93 kcal/mole, -161.16 kcal/mole, and - 151.44 kcal/mole, respectively. These energies suggested that P3 is strongly bound to the active site of NADH-dependent enoyl-acyl carrier protein reductase. Overall, this study proposed the application of these peptides as a possible therapeutic solution to inhibit the growth of Mycobacterium tuberculosis.

Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo.

Inflammation plays a crucial role in the development and progression of many diseases, and is often caused by dysregulation of signalling from pattern recognition receptors, such as TLRs. Inhibition of key protein-protein interactions is an attractive target for treating inflammation. Recently, we demonstrated that the signalling lymphocyte activation molecule family 1 (SLAMF1) positively regulates signalling downstream of TLR4 and identified the interaction interface between SLAMF1 and the TLR4 adaptor protein TRIF-related adapter molecule (TRAM). Based on these findings, we developed a SLAMF1-derived peptide, P7, which is linked to a cell-penetrating peptide for intracellular delivery. We found that P7 peptide inhibits the expression and secretion of IFNβ and pro-inflammatory cytokines (TNF, IL-1β, IL-6) induced by TLR4, and prevents death in mice subjected to LPS shock. The mechanism of action of P7 peptide is based on interference with several intracellular protein-protein interactions, including TRAM-SLAMF1, TRAM-Rab11FIP2, and TIRAP-MyD88 interactions. Overall, P7 peptide has a unique mode of action and demonstrates high efficacy in inhibiting TLR4-mediated signalling in vitro and in vivo.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.

Antibody reactivity to Mycobacterium tuberculosis-specific regions of differences 1 and regions of differences 9 proteins and peptides in rabbits, mice, and humans.

The major antigens encoded by Mycobacterium tuberculosis-specific genomic regions of differences (RDs) could be useful in the development of new vaccines and/or diagnostic reagents using T-cell and/or antibody assays. In particular, RD1 proteins PE35, PPE68, ESXA, ESXB, and RD9 protein ESXV and their peptides have been identified as major T-cell antigens. However, little is known about their antibody reactivities in different mammalian species. This study aims to determine the antigen-specific antibody reactivities to the above antigens and their peptides in three different mammalian species, i.e., rabbits, mice, and humans. Sera were obtained from (i) rabbits immunized with purified recombinant proteins PE35, PPE68, ESXA, ESXB, and ESXV; (ii) mice immunized with recombinant DNA vaccine constructs of pUMVC6 and pUMVC7 containing RD1 and RD9 genes; and (iii) tuberculosis (TB) patients and healthy humans. Enzyme-linked immunosorbent assays (ELISAs) were performed with the sera to determine the antibody reactivity to purified recombinant proteins, peptide pools, and individual peptides of RD1 and RD9 proteins. The ELISA results with sera from rabbits immunized with pure recombinant proteins showed positive antibody reactivity with all of the immunizing proteins and their synthetic peptide pools. Testing of the sera with individual peptides showed positive antibody reactivity with PE35 peptides P1 (aa 1-25), P2 (aa 16-40), P5 (aa 61-85), and P6 (aa 76-99); PPE68 peptides P9 (aa 121-145), P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371); all peptides (P1 to P6) of ESXA and ESXB; and ESXV peptides P1 (aa 1-25), P2 (aa 16-40), P3 (aa 31-55), P5 (aa 61-85), and P6 (aa 76-94). The sera from mice immunized with DNA vaccine constructs showed antibody reactivity to all proteins and the peptide P6 (aa 76-99) of PE35 and peptides P19 (aa 271-295) and P24 (aa 346-371) of PPE68. In humans, the peptides P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371) of PPE68 and the peptides P4 (aa 46-70), P5 (aa 61-85), and P6 (aa 76-94) of ESXV showed positive reactivity with sera from TB patients and healthy controls. The results demonstrate the presence of several antibody epitopes in each protein, but variations in the epitopes recognized were observed among mice, rabbits, and humans, which could be due to mammalian species differences and/or mode of antigen delivery.

Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 μg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 μg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 μg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.

Targeted protein degradation (TPD) confers knockdown of "undruggable" targets such as alpha-synuclein (αSyn), a pathogenic protein in multiple neurodegenerative diseases. Though many of these proteins were mainly degraded through the autophagy-lysosome pathway (ALP), few TPD tools harnessing the ALP were reported. Herein, we developed a strategy termed autophagosome-anchoring chimera (ATACC), in which the protein of interest (POI) can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of an LC3-interacting region (LIR)-containing bifunctional peptide, and the selective autophagy of the POI is thus facilitated. A series of αSyn-targeting ATACC peptides were designed and synthesized. Biological evaluations demonstrated that these compounds could degrade αSyn specifically and effectively through a "chemical-induced cargo recognition-ALP degradation" mechanism. The neuroprotective effects of ATACC peptide P1 were further validated in vitro and in vivo. Collectively, our results provided a new TPD tool and revealed a potential therapeutic strategy against synucleinopathies.

An electrochemical biosensor based on graphene intercalated functionalized black phosphorus/gold nanoparticles nanocomposites for the detection of bacterial enzyme

Sortase A (Srt A) is a key enzyme that catalyzes the covalent anchoring of surface proteins to the bacterial cell wall and plays a dominant role in the late stage of Gram-positive bacterial infections. Achieving activity detection of Srt A and inhibitors screening is important for the treatment of Gram-positive bacterial infections and novel antibacterial drugs research. Here, an electrochemical sensor pattern is proposed. Black phosphorus (BP) is a novel graphite-like two-dimensional material with excellent electrochemical properties. However, the extremely poor environmental stability limits its practical application. The surface modification of BP using graphene oxide (GO) significantly improves the stability, and obtained partially reduced graphite oxide functionalized BP composites combine both electrical conductivity and stability. In this paper, we develop an electrochemical biosensor based on partially reduced graphite oxide functionalized BP /gold nanoparticles (p-rGO-BP/AuNPs) nanocomposites for detecting Srt A activity. The assay utilizes two peptide chains, P1 (CLPETG) and P2 (GGGGG), which can be linked by the presence of Srt A. At the same time, the process is monitored by differential pulse voltammetry (DPV). The sensor showed good selectivity and stability with a linear dynamic range of 1 pM-100 nM and a detection limit (LOD) of 0.65 pM. In addition, the method was successfully used to detect Srt A in complex samples and estimate the half-inhibitory concentration (IC50) of Srt A inhibitors (curcumin, dihydromyricetin and rutin).

A Metalloproteinase Cocktail from the Venom of Protobothrops flavoviridis Cleaves Amyloid Beta Peptides at the α-Cleavage Site.

A disintegrin and metalloproteinase (ADAM) family proteins are a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell surface protein ectodomains, including amyloid precursor protein (APP). Human ADAM 9, 10, and 17 proteolyze APPs and produce non-amyloid-genic p3 peptides, instead of neurotoxic amyloid-β peptides (Aβs; Aβ40 and Aβ42), which form fibrils and accumulate in the brain of patients with Alzheimer's disease (AD). The ADAM family is closely related to snake venom metalloproteinases (SVMPs), which are derived from ancestral ADAMs but act as soluble proteinases. To test the therapeutic potential of SVMPs, we purified SVMPs from Protobothrops flavoviridis venom using metal ion affinity and pooled into a cocktail. Thus, 9 out of 11 SVMPs in the P. flavoviridis genome were identified in the cocktail. SVMPs inhibited Aβ secretion when added to human cell culture medium without affecting APP proteolysis. SVMPs degraded synthetic Aβ40 and Aβ42 peptides at the same cleavage site (α-site of APP) as ADAM9, 10, and 17. SVMPs did not degrade Aβ fibrils but interfered with their formation, assessed using thioflavin-T. Thus, SVMPs have therapeutic potential for AD as an Aβ-degrading protease, and the finding adds to the discovery of bioactive peptides from venoms as novel therapeutics.

Vectorization via Siderophores Increases Antibacterial Activity of K(RW)3 Peptides against Pseudomonas aeruginosa.

A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore-type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well-established arginine-tryptophane combination K(RW)3 (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti-bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a-c) ; (iii) the non-siderophore chelators deferasirox (DFX) and deferiprone-carboxylate (DFP-COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR-MS). 55 Fe uptake assays indicate a receptor-mediated uptake for 4 a-c, DFP-COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron-depleted medium. MIC values were as low as 2 μM (for P1-DFP) on wild type P. aeruginosa. The activity of P1-DFO and P1-DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 μM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by 55 Fe uptake studies, the P1-DFX conjugate had excellent antibacterial activity compared to P1 (2 μM, and as low as 0.25 μM on a receptor-deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1-DFP and P1-DFX being the most potent conjugates.

Nanodroplet-Based Reagent Delivery into Water-in-Fluorinated-Oil Droplets.

In vitro compartmentalization (IVC) is a technique for generating water-in-oil microdroplets to establish the genotype (DNA information)-phenotype (biomolecule function) linkage required by many biological applications. Recently, fluorinated oils have become more widely used for making microdroplets due to their better biocompatibility. However, it is difficult to perform multi-step reactions requiring the addition of reagents in water-in-fluorinated-oil microdroplets. On-chip droplet manipulation is usually used for such purposes, but it may encounter some technical issues such as low throughput or time delay of reagent delivery into different microdroplets. Hence, to overcome the above issues, we demonstrated a nanodroplet-based approach for the delivery of copper ions and middle-sized peptide molecules (human p53 peptide, 2 kDa). We confirmed the ion delivery by microscopic inspection of crystal formation inside the microdroplet, and confirmed the peptide delivery using a fluorescent immunosensor. We believe that this nanodroplet-based delivery method is a promising approach to achieving precise control for a broad range of fluorocarbon IVC-based biological applications, including molecular evolution, cell factory engineering, digital nucleic acid detection, or drug screening.

Computational peptide engineering approach for selection of the new C05 antibody-driven peptide with potency to blocking influenza a virus attachment; from in silico to in vivo

Antiviral drugs are currently used to prevent or treat viral infections like influenza A Virus (IAV). Nonetheless, annual genetic mutations of influenza viruses make them resistant to efficient treatment by current medications. Antiviral peptides have recently attracted researchers’ attention and can potentially supplant the current medications. This study aimed to design peptides against IAV propagation. For this purpose, P2 and P3 peptides were computationally designed based on the HCDR3 region of the C05 antibody (a monoclonal antibody that neutralizes influenza HA protein and inhibits the virus attachment). The synthesized peptides were tested against the influenza A virus (A/Puerto Rico/8/34 (H1N1)) in vitro, and the most efficient peptide was selected for in vivo experiments. It was shown that the designed peptide shows much more prophylactic and therapeutic effects against the virus. These findings demonstrated that the designed peptide can control the virus infection without any cytotoxicity effect. Antiviral peptide design is acknowledged as a critical tactic to manage viral infections by preventing viral binding to the host cells. Communicated by Ramaswamy H. Sarma

Novel Antimicrobial Peptides from Saline Environments Active against E. faecalis and S. aureus: Identification, Characterisation and Potential Usage.

Microorganisms inhabiting saline environments have been known for decades as producers of many valuable bioproducts. These substances include antimicrobial peptides (AMPs), the most recognizable of which are halocins produced by halophilic Archaea. As agents with a different modes of action from that of most conventionally used antibiotics, usually associated with an increase in the permeability of the cell membrane as a result of a formation of channels and pores, AMPs are a currently promising object of research focused on the investigation of antibiotics with non-standard modes of action. The aim of this study was to investigate antimicrobial activity against multidrug-resistant human pathogens of three peptides, which were synthetised based on sequences identified in metagenomes from saline environments. The investigations were performed against Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Subsequently, the cytotoxicity and haemolytic properties of the tested peptides were verified. An in silico analysis of the interaction of the tested peptides with molecular targets for reference antibiotics was also carried out in order to verify whether or not they can act in a similar way. The P1 peptide manifested the growth inhibition of E. faecalis at a MIC50 of 32 µg/mL and the P3 peptide at a MIC50 of 32 µg/mL was shown to inhibit the growth of both E. faecalis and S. aureus. Furthermore, the P1 and P3 peptides were shown to have no cytotoxic or haemolytic activity against human cells.

Dual-targeting AAV9P1-mediated neuronal reprogramming in a mouse model of traumatic brain injury.

Traumatic brain injury results in neuronal loss and glial scar formation. Replenishing neurons and eliminating the consequences of glial scar formation are essential for treating traumatic brain injury. Neuronal reprogramming is a promising strategy to convert glial scars to neural tissue. However, previous studies have reported inconsistent results. In this study, an AAV9P1 vector incorporating an astrocyte-targeting P1 peptide and glial fibrillary acidic protein promoter was used to achieve dual-targeting of astrocytes and the glial scar while minimizing off-target effects. The results demonstrate that AAV9P1 provides high selectivity of astrocytes and reactive astrocytes. Moreover, neuronal reprogramming was induced by downregulating the polypyrimidine tract-binding protein 1 gene via systemic administration of AAV9P1 in a mouse model of traumatic brain injury. In summary, this approach provides an improved gene delivery vehicle to study neuronal programming and evidence of its applications for traumatic brain injury.

In vitro and in silico studies for the identification of anti-cancer and antibacterial peptides from camel milk protein hydrolysates.

Today, breast cancer and infectious diseases are very worrying that led to a widespread effort by researchers to discover natural remedies with no side effects to fight them. In the present study, we isolated camel milk protein fractions, casein and whey proteins, and hydrolyzed them using pepsin, trypsin, and both enzymes. Screening of peptides with anti-breast cancer and antibacterial activity against pathogens was performed. Peptides derived from whey protein fraction with the use of both enzymes showed very good activity against MCF-7 breast cancer with cell viability of 7.13%. The separate use of trypsin and pepsin to digest whey protein fraction yielded peptides with high antibacterial activity against S. aureus (inhibition zone of 4.17 ± 0.30 and 4.23 ± 0.32 cm, respectively) and E. coli (inhibition zone of 4.03 ± 0.15 and 4.03 ± 0.05 cm, respectively). Notably, in order to identify the effective peptides in camel milk, its protein sequences were retrieved and enzymatically digested in silico. Peptides that showed both anticancer and antibacterial properties and the highest stability in intestinal conditions were selected for the next step. Molecular interaction analysis was performed on specific receptors associated with breast cancer and/or antibacterial activity using molecular docking. The results showed that P3 (WNHIKRYF) and P5 (WSVGH) peptides had low binding energy and inhibition constant so that they specifically occupied active sites of protein targets. Our results introduced two peptide-drug candidates and new natural food additive that can be delivered to further animal and clinical trials.

Synthesis, Characterization, and Antimicrobial Activity of Ultra-Short Cationic β-Peptides.

The development of new antibiotics is urgently required because of the rapidly growing resistance against conventional antibiotics. The antimicrobial peptides show potential as small antibiotic molecules. The stability of peptides is a primary concern for the use of peptides as drugs. Introducing β-amino acids into peptide sequences can be useful in preventing biological degradation by proteolytic enzymes. Herein, we describe the synthesis, characterization, and antimicrobial activity of ultra-short cationic β-peptides, LA-β3,3-Pip-β2,2-Ac6c-PEA, P1; LA-β3,3-Pip(G)-β2,2-Ac6c-PEA, P2; LAU-β3,3-Pip-β2,2-Ac6c-PEA, P3, and LAU-β3,3-Pip(G)-β2,2-Ac6c-PEA, P4. Peptides P1-P4 were evaluated against Gram-negative, Gram-positive, MRSA, and multi-drug resistant E. coli (MDR-E. coli). P3 exhibited the most potent antimicrobial activity against E. coli, S. epidermidis, S. aureus, K. pneumoniae, S. mutans, and E. faecalis, with MIC values 0.5, 2, 0.5, 1, 2, and 1 μg/mL, respectively. P3 exhibited time- and concentration-dependent bactericidal activities against E. coli, S. aureus, and E. faecalis with a killing rate of 1.6 logs/h. The treatment of E. coli with peptide P3 showed membrane disruption. In addition, P3 exhibited the inhibition of biofilm produced by E. coli, synergism with antibiotics (ciprofloxacin, streptomycin, and ampicillin), 100% cell viability against AML12, RAW 264.7, and HEK-293 cell lines at 1, and 10 μg/mL concentrations.