Structure-guided design and cloning of peptide inhibitors targeting CDK9/cyclin T1 protein-protein interaction.

CDK9 (cyclin-dependent kinase 9) plays a significant role in numerous pathological conditions, such as HIV-1 infection and cancer. The interaction between CDK9 and cyclin T1 is crucial for maintaining the kinase's active state. Therefore, targeting this protein-protein interaction offers a promising strategy for inhibiting CDK9. In this study, we aimed to design and characterize a library of mutant peptides based on the binding region of cyclin T1 to CDK9. Using Osprey software, a total of 7,776 mutant peptides were generated. After conducting a comprehensive analysis, three peptides, namely, mp3 (RAADVEGQRKRRE), mp20 (RAATVEGQRKRRE), and mp29 (RAADVEGQDKRRE), were identified as promising inhibitors that possess the ability to bind to CDK9 with high affinity and exhibit low free binding energy. These peptides exhibited favorable safety profiles and displayed promising dynamic behaviors. Notably, our findings revealed that the mp3 and mp29 peptides interacted with a conserved sequence in CDK9 (residues 60-66). In addition, by designing the structure of potential peptides in the plasmid vector pET28a (+), we have been able to pave the way for facilitating the process of their recombinant production in an Escherichia coli expression system in future studies. Predictions indicated good solubility upon overexpression, further supporting their potential for downstream applications. While these results demonstrate the promise of the designed peptides as blockers of CDK9 with high affinity, additional experimental studies are required to validate their biological activity and assess their selectivity. Such investigations will provide valuable insights into their therapeutic potential and pave the way for the future development of peptide-based inhibitors targeting the CDK9-cyclin T1 complex.

Virus-like particles (VLPs): A promising platform for combating against Newcastle disease virus.

The global poultry industry plays a pivotal role in providing eggs and meat for human consumption. However, outbreaks of viral disease, especially Newcastle virus disease (NDV), within poultry farms have detrimental effects on various zootechnical parameters, such as body weight gain, feed intake, feed conversion ratio, as well as the quality of egg and meat production. Cases of vaccine failure have been reported in regions where highly pathogenic strains of NDV are prevalent. To tackle this challenge, virus-like particles (VLPs) have emerged as a potential solution. VLPs closely resemble natural viruses, offering biocompatibility and immune-stimulating properties that make them highly promising for therapeutic applications against NDV. Hence, this review emphasizes the significance of NDV and the need for effective treatments. The manuscript will contain several key aspects, starting with an exploration of the structure and properties of NDV. Subsequently, the paper will delve into the characteristics and benefits of VLPs compared to conventional drug delivery systems. A comprehensive analysis of VLPs as potential vaccine candidates targeting NDV will be presented, along with a discussion on strategies for loading cargo into these NDV-targeting VLPs. The review will also examine various expression systems utilized in the production of NDV-targeting VLPs. Additionally, the manuscript will address future prospects and challenges in the field, concluding with recommendations for further research.

Strategies for oral delivery of bioactive peptides with focus on debittering and masking.

Protein hydrolysis is a process used in the food industry to generate bioactive peptides of low molecular weight and with additional health benefits, such as antihypertensive, antidiabetic, and antioxidant properties that are often associated with their content on hydrophobic amino acids. This results in an increased bitterness of the products, making them less desirable for their use in food formulations. This review summarizes the main dietary sources of bitter bioactive peptides, including methods to determine their bitterness, such as the Q-values and electronic tongue; and the main factors and mechanisms underlying the bitterness of these compounds. The main strategies currently used to improve the taste and oral delivery of bioactive peptides are also discussed together with the main advantages and drawbacks of each technique. Debittering and masking techniques are reported in detail, including active carbon treatments, alcohol extraction, isoelectric precipitation, chromatographic methods, and additional hydrolytic processes. Other masking or blocking techniques, including the use of inhibitors, such as modified starch, taurine, glycine, and polyphosphates, as well as chemical modifications, such as amination, deamination, acetylation, or cross-linking were also discussed. The findings of this work highlight encapsulation as a highly effective method for masking the bitter taste and promoting the bioactivity of peptides compared to other traditional debittering and masking processes. In conclusion, the article suggests that advanced encapsulation technologies can serve as an effective means to mitigate the bitterness associated with bioactive peptides, while simultaneously preserving their biological activity, increasing their viability in the development of functional foods and pharmaceuticals.

System network analysis of Rosmarinus officinalis transcriptome and metabolome-Key genes in biosynthesis of secondary metabolites.

Medicinal plants contain valuable compounds that have attracted worldwide interest for their use in the production of natural drugs. The presence of compounds such as rosmarinic acid, carnosic acid, and carnosol in Rosmarinus officinalis has made it a plant with unique therapeutic effects. The identification and regulation of the biosynthetic pathways and genes will enable the large-scale production of these compounds. Hence, we studied the correlation between the genes involved in biosynthesis of the secondary metabolites in R. officinalis using proteomics and metabolomics data by WGCNA. We identified three modules as having the highest potential for the metabolite engineering. Moreover, the hub genes highly connected to particular modules, TFs, PKs, and transporters were identified. The TFs of MYB, C3H, HB, and C2H2 were the most likely candidates associated with the target metabolic pathways. The results indicated that the hub genes including Copalyl diphosphate synthase (CDS), Phenylalanine ammonia lyase (PAL), Cineole synthase (CIN), Rosmarinic acid synthase (RAS), Tyrosine aminotransferase (TAT), Cinnamate 4-hydroxylase (C4H), and MYB58 are responsible for biosynthesis of important secondary metabolites. Thus, we confirmed these results using qRT-PCR after treating R. officinalis seedlings with methyl jasmonate. These candidate genes may be employed for genetic and metabolic engineering research to increase R. officinalis metabolite production.

Impact of Whey Protein Edible Coating Containing Fish Gelatin Hydrolysates on Physicochemical, Microbial, and Sensory Properties of Chicken Breast Fillets.

This study aims to research the impact of coatings containing whey protein (WP), fish gelatin hydrolysates (FGH), and both compounds together (WP + FGH) on the shelf-life of chicken breast fillets over the course of 16 days of cold storage (4 °C, 4-day intervals), as assessed by their physicochemical, microbiological, and sensory properties. Overall, cooking loss, pH value, total volatile base nitrogen, free fatty acids, peroxide value, and thiobarbituric acid reactive substances increased with storage time in all samples. WP + FGH coated samples had significantly lower variation in all these parameters over the time of storage compared to other coated samples (WP and FGH), while these parameters increased greatly in control (uncoated) samples. WP + FGH coating also resulted in reduced bacterial counts of total mesophilic, aerobic psychrotrophic, and lactic acid bacteria compared to other coated and uncoated samples. The sensory evaluation revealed no differences in the panelists' overall acceptance at day 0 of storage between samples. The samples were considered "non-acceptable" by day 8 of storage; however, WP + FGH coated samples maintained an overall higher acceptability score for the sensory attributes evaluated by the panelists. Overall, this study shows the potential of WP + FGH coatings for prolonging the shelf-life of chicken breast fillets.

Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor.

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

Experimental, molecular docking and molecular dynamic studies of natural products targeting overexpressed receptors in breast cancer.

Natural compounds are proper tools for inhibiting cancer cell proliferation. Hence, the search for these ligands of overexpressed receptors in breast cancer has been a competitive challenge recently and opens new avenues for drug discovery. In this research, we have investigated molecular interactions between natural products and overexpressed receptors in breast cancer using molecular docking and dynamic simulation approaches followed by extraction of the best ligand from Citrus limetta and developing for nanoscale encapsulation composed of soy lecithin using a sonicator machine. The encapsulation process was confirmed by DLS and TEM analyses. Anticancer activity was also examined using MTT method. Among the investigated natural compounds, hesperidin was found to bind to specific targets with stronger binding energy. The molecular dynamics results indicated that the hesperidin-MCL-1 complex is very stable at 310.15 K for 200 ns. The RP-HPLC analysis revealed that the purity of extracted hesperidin was 98.8% with a yield of 1.72%. The results of DLS and TEM showed a strong interaction between hesperidin and lecithin with an entrapped efficiency of 92.02 ± 1.08%. Finally, the cytotoxicity effect of hesperidin was increased against the MDA-MB-231 cell line with an IC50 value of 62.93 µg/mL after encapsulation, whereas no significant effect against the MCF10A cell line. We showed for the first time that hesperidin is a flexible and strong ligand for the MCL-1 receptor. Also, it has the in vitro ability to kill the MDA-MB-231 cell lines without having a significant effect on the MCF10A cell lines. Therefore, hesperidin could be used as a food ingredient to generate functional foods.

Successful use of Nicotiana tabacum hairy roots for the recombinant production of Cecropin A peptide.

Cecropin A, as an antimicrobial peptide (AMP), is possible to use in medical and agricultural fields as a new and safe biocontrol agent. Therefore, it is highly necessary to find a cost-effective and scalable approach to generate a large scale of it. In this research, the Agrobacterium rhizogenes strain ATCC 15834 was used to transfer the Cecropin A gene to the Nicotiana tabacum. After confirmation of transgenic hairy roots, the antibacterial activity of purified Cecropin A peptide was measured using the agar gel diffusion method. Successful transforming of Cecropin A was confirmed at the RNA and protein levels in hairy root cells using RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The highest Cecropin A amount was detected in line 4 of the transgenic lines using ELISA in comparison with the nontransgenic line. Subsequently, the antimicrobial activity of Cecropin A extracted from line 4 showed the highest inhibition activity against Aspergillus niger. Besides, this activity was stable against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans pathogens after 7 days. The recombinant production of Cecropin A AMP had a yield of 63.81 µg/g of fresh weight. According to a significant yield, this system can be used to produce the Cecropin A peptide for pharmacological and food science applications.

Novel bioactive peptides of Achillea eriophora show anticancer and antioxidant activities.

Despite the limitations of current methods in cancer treatment, the use of bioactive peptides can be as an alternative to treat today. Therefore, isolation and relative purification of bioactive peptides was carried out form Achillea eriophora using a Sep-Pak C18 SPE cartridge and Amicon® Ultra Centrifugal Filters. The presence of desired peptides was checked using RP-HPLC and confirmed using LC-MS. The results of anticancer assay showed that the peptide mixture inhibits the growth of MCF-7 cancerous cell line with the values of IC50, GI50, and LC50 equal to 18.73 ± 0.22, 7.52 ± 0.15, and 56.73 ± 0.18 µg/mL, respectively. It also showed DPPH radical scavenging activity and cupric-ion reducing power with the IC50 value of 5.095 ± 0.23 and 63.3 ± 0.44 µg/mL, respectively. Although flavonoids were present in the sample along with the peptides, their amount was trivial (18.097 ± 1.36 µg/mL). Nevertheless, the results of the LC-MS showed mass-to-charge ratios of 301.17, 261.22, and 243.25, which was a dipeptide or tripeptide in compression to enzyme-digested BSA as a standard. In addition, SEM analysis of the purified peptide mixture showed that it kills the MCF-7 cancerous cell line by creating pores in the membrane. Therefore, it might be valuable to these peptides sequenced and be studied for physicochemical properties. Animal and clinical studies could help its application in drug development.

The potential application of the protein hydrolysates of three medicinal plants: cytotoxicity and functional properties.

Functional evaluation of encrypted bioactive peptides in protein structure helps to better understand those for using in pharmacy and food sciences. For this purpose, the total protein was extracted from Matricaria chamomilla, Ziziphora clinopodioides, and Cressa cretica, and partially purified with ammonium sulfate. Protein hydrolysates were obtained from pancreatin hydrolysis for 240 min and the enzyme hydrolysis was confirmed using the determination of hydrolysis degree and Fourier transform infrared (FT-IR) followed by the physicochemical and sensory properties were investigated. The results showed that all hydrolysates had both cytotoxic and antioxidant activities. Specifically, C. cretica hydrolysates represented cytotoxic activity against the MCF-7 cell line with the IC50 of 135.21 µg/mL, while showed no significant growth inhibition effect on the HEK293 cell line. Besides, M. chamomilla hydrolysates showed the lowest bitterness value (1.125 ± 0.52). From the perspective of color investigation, M. chamomilla hydrolysates indicated the highest L* and the lowest a* factors. The highest turbidity and surface tension, and 10-fold more cancer cell killing effect under gastrointestinal digestion conditions were observed for M. chamomilla hydrolysates. Therefore, bioactive peptides might be formulated in designing of novel anticancer drugs or could be used in promising protocols for the production of food products with beneficial health effects.

Physicochemical, rheological, and molecular characterization of colloidal gelatin produced from Common carp by-products using microwave and ultrasound-assisted extraction.

The effects of ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) methods on molecular and physicochemical characteristics of the resultant gelatin were examined. Before extraction procedure, we investigated the optimum pH for swelling of Common carp by-products, which is an important pretreatment for gelatin production. The highest swelling yield was achieved at pH 13 among pH 1-14 with unit intervals. Results indicated that the UAE gelatin has a higher gel strength, viscosity, melting point, and gelling point. The power and time of sonication showed a reverse relation with these characteristics. In addition, as the time of microwave heating was raised, the gel strength, viscosity, melting point, and gelling point were decreased. The FT-IR spectra showed similar peaks but the Amide B in UAE gelatin slightly vanished. The electrophoretic pattern also revealed the higher gel strength and viscosity of UAE gelatin due to the higher intensity of α and ß chains compared to MAE gelatin. It can be concluded from all of the results of this study that the produced gelatin using these procedures can be a good source of gelatin in food and drug industries.