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Abstract: The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNALys3 UUU as the primer for reverse transcription. NCp7 also remodels the htRNA''s amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA''s unique composition of post-transcriptional modifications on NCp7 recognition of htRNALys3 UUU, the protein''s binding and functional remodeling of the human anticodon stem and loop domain (hASLLys3) were studied. NCp7 bound the hASLLys3 UUU modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm5s2U34) and 2-methylthio-N 6 -threonylcarbamoyladenosine at position-37 (ms2t6A37) with a considerably higher affinity than the unmodified hASLLys3 UUU (K d =0.28±0.03 and 2.30±0.62 μM, respectively). NCp7 denatured the structure of the hASLLys3 UUU-mcm5s2U34;ms2t6A37;Ψ39 more effectively than that of the unmodified hASLLys3 UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K d =0.55±0.10 μM) and specificity for the ASLLys3 UUU-mcm5s2U34;ms2t6A37 comparable to that of NCp7. The peptides recognized a t6A37-modified ASL with an affinity (K d =0.60±0.09 μM) comparable to that for hASLLys3 UUU-mcm5s2U34;ms2t6A37, indicating a preference for the t6A37 modification. Significantly, one of the peptides was capable of relaxing the hASLLys3 UUU-mcm5s2U34;ms2t6A37;Ψ39 structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNALys3 UUU have been found to be important determinants of NCp7''s recognition prior to the tRNALys3 UUU being annealed to the viral genome as the primer of reverse transcription. [Copyright &y& Elsevier]

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS Sap I restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential. • A break-through vector-enzymatic DNA fragment amplification-expression technology for construction of concatemeric DNA, RNA and proteins is presented. • Concatemers are ready for genetic expression and can contain hundreds of copies of monomers arranged in ordered, head-to-tail fasion. • The technology for construction of novel biomaterials has numerous applications: biomedical, industrial, research and environmental. • The technology has already being practically applied, is covered by issued patent no PL 228341-B1 as well as pending worldwide patent applications. [ABSTRACT FROM AUTHOR]

Adipose-derived mesenchymal stromal cells (AD-MSCs) are an essential issue in modern medicine. Extensive preclinical and clinical studies have shown that mesenchymal stromal/stem cells, including AD-MSCs, have specific properties (ability to differentiate into other cells, recruitment to the site of injury) of particular importance in the regenerative process. Ongoing research aims to elucidate factors supporting AD-MSC culture and differentiation in vitro. Angiopoietin-like proteins (ANGPTLs), known for their pleiotropic effects in lipid and glucose metabolism, may play a significant role in this context. Regeneration is a complex and dynamic process controlled by many factors. ANGPTL6 (Angiopoietin-related growth factor, AGF), among many activities modulated the biological activity of stem cells. This study examined the influence of synthesized AGF-derived peptides, designated as AGF9 and AGF27, on AD-MSCs. AGF9 and AGF27 enhanced the viability and migration of AD-MSCs and acted as a chemotactic factor for these cells. AGF9 stimulated chondrogenesis and lipid synthesis during AD-MSCs differentiation, influenced AD-MSCs cytokine secretion and modulated transcriptome for such basic cell activities as migration, transport of molecules, and apoptosis. The ability of AGF9 to modulate the biological activity of AD-MSCs warrants the consideration of this peptide a noteworthy therapeutic agent that deserves further investigation for applications in regenerative medicine., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M. Pikula, P. Sachadyn, A. Piotrowski reports financial support was provided by National Centre for Research and Development. P. Mucha, I. Puchalska, M. Pikula, A. Schumacher, M. Cichorek, A. Wardowska, M. Deptula, A. Piotrowski, N. Filipowicz, P. Sachadyn has patent #Patent number: PL236332 B1, 28 Dec 2020 issued to assignee. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins., Results: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected., Conclusions: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties., (© 2024. The Author(s).)

Nucleopeptides (NPs) represent synthetic polymers created by attaching nucleobases to the side chains of amino acid residues within peptides. These compounds amalgamate the characteristics of peptides and nucleic acids, showcasing a unique ability to recognize RNA structures. In this study, we present the design and synthesis of Fmoc-protected nucleobase amino acids (1,4-TzlNBAs) and a new class of NPs, where canonical nucleobases are affixed to the side chain of L -homoalanine (Hal) through a 1,4-linked-1,2,3-triazole (HalTzl). Fmoc-protected 1,4-TzlNBAs suitable for HalTzl synthesis were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation of Fmoc- L -azidohomoalanine (Fmoc-Aha) and N1- or N9 -propargylated nucleobases or their derivatives. Following this, two trinucleopeptides, HalTzl AAA and HalTzl AGA , and the hexanucleopeptide HalTzl TCCCAG , designed to complement bulge and outer loop structures of TAR ( trans -activation response element) RNA HIV-1, were synthesized using the classical solid-phase peptide synthesis (SPPS) protocol. The binding between HalTzls and fluorescently labeled 5'-(FAM(6))-TAR UCU and UUU mutant was characterized using circular dichroism (CD) and fluorescence spectroscopy. CD results confirmed the binding of HalTzls to TAR RNA, which was evident by a decrease in ellipticity band intensity around 265 nm during complexation. CD thermal denaturation studies indicated a relatively modest effect of complexation on the stability of TAR RNA structure. The binding of HalTzls at an equimolar ratio only marginally increased the melting temperature (T m ) of the TAR RNA structure, with an increment of less than 2 °C in most cases. Fluorescence spectroscopy revealed that HalTzl AAA and HalTzl AGA , complementary to UUU or UCU bulges, respectively, exhibited disparate affinities for the TAR RNA structure (with K d ≈ 30 and 256 µM, respectively). Hexamer HalTzl TCCCAG , binding to the outer loop of TAR UCU , demonstrated a moderate affinity with K d ≈ 38 µM. This study demonstrates that newly designed HalTzls effectively bind the TAR RNA structure, presenting a potential new class of RNA binders and may be a promising scaffold for the development of a new class of antiviral drugs.

Arginine-rich cell-penetrating peptides (RRCPPs) exhibit intrinsic neuroprotective effects on neurons injured by acute ischemic stroke. Conformational properties, interaction, and the ability to penetrate the neural membrane are critical for the neuroprotective effects of RRCCPs. In this study, we applied circular dichroism (CD) spectroscopy and coarse-grained molecular dynamics (CG MD) simulations to investigate the interactions of two RRCPPs, Tat(49-57)-NH 2 (arginine-rich motif of Tat HIV-1 protein) and PTD4 (a less basic Ala-scan analog of the Tat peptide), with an artificial neuronal membrane (ANM). CD spectra showed that in an aqueous environment, such as phosphate-buffered saline, the peptides mostly adopted a random coil (PTD4) or a polyproline type II helical (Tat(49-57)-NH 2 ) conformation. On the other hand, in the hydrophobic environment of the ANM liposomes, the peptides showed moderate conformational changes, especially around 200 nm, as indicated by CD curves. The changes induced by the liposomes were slightly more significant in the PTD4 peptide. However, the nature of the conformational changes could not be clearly defined. CG MD simulations showed that the peptides are quickly attracted to the neuronal lipid bilayer and bind preferentially to monosialotetrahexosylganglioside (DPG1) molecules. However, the peptides did not penetrate the membrane even at increasing concentrations. This suggests that the energy barrier required to break the strong peptide-lipid electrostatic interactions was not exceeded in the simulated models. The obtained results show a correlation between the potential of mean force parameter and a peptide's cell membrane-penetrating ability and neuroprotective properties.

Contact tracing and quarantine are well established non-pharmaceutical epidemic control tools. The paper aims to clarify the impact of these measures in evolution of epidemic. The proposed deterministic model defines a simple rule on the reproduction number [Formula: see text] in terms of ratio of diagnosed cases and, quarantine and transmission parameters. The model is applied to the early stage of Covid19 crisis in Poland. We investigate 3 scenarios corresponding to different ratios of diagnosed cases. Our results show that, depending on the scenario, contact tracing prevented from 50% to over 90% of cases. The effects of quarantine are limited by fraction of undiagnosed cases. The key conclusion is that under realistic assumptions the epidemic can not be controlled without any social distancing measures., Competing Interests: The authors have declared that no competing interests exist.

Applications of bioactive peptides and polypeptides are emerging in areas such as drug development and drug delivery systems. These compounds are bioactive, biocompatible and represent a wide range of chemical properties, enabling further adjustments of obtained biomaterials. However, delivering large quantities of peptide derivatives is still challenging. Several methods have been developed for the production of concatemers - multiple copies of the desired protein segments. We have presented an efficient method for the production of peptides of desired length, expressed from concatemeric Open Reading Frame. The method employs specific amplification-expression DNA vectors. The main methodological approaches are described by Skowron et al., 2020 [1]. As an illustration of the demonstrated method's utility, an epitope from the S protein of Hepatitis B virus (HBV) was amplified. Additionally, peptides, showing potentially pro-regenerative properties, derived from the angiopoietin-related growth factor (AGF) were designed and amplified. Here we present a dataset including: ( i ) detailed protocols for the purification of HBV and AGF - derived polyepitopic protein concatemers, ( ii ) sequences of the designed primers, vectors and recombinant constructs, ( iii ) data on cytotoxicity, immunogenicity and stability of AGF-derived polypeptides., (© 2019 The Author(s).)

Background: Most studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity., Methods: We applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice., Findings: We observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2 ± 9.4% in zebularine-treated and by 43.6 ± 15.4% in control mice (p < 10 -30 ). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues., Interpretation: This study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.