Small secreted peptides (SSPs) play an important role in modulating immune responses in all land plants. However, the evolution of stress peptide signaling in different plant phyla remains poorly understood. Here, we compared the expression of SSP genes in the pathogen-induced transcriptomes of vascular and non-vascular plants. We found 13, 19, 15, and 28 SSP families that were differentially expressed during infection in Physcomitrium patens, Zea mays, Brassica napus, and Solanum tuberosum, respectively. A comparative study of peptide motifs and predicted three-dimensional structures confirmed the similarity of SSPs across the examined plant species. In both vascular and non-vascular plants. However, only the RALF peptide family was differentially regulated under infection. We also found that EPFL peptides, which are involved in growth and development processes in angiosperms, were differentially regulated in P. patens in response to pathogen infection. The search for novel immune-specific peptides revealed a family of PSY-like peptides that are differentially regulated during infection in P. patens. The treatment with synthetic tyrosine-modified and non-modified PSY, and PSY-like peptides, as well as recombinant EPFL and MEG, validated their roles in the immune response and growth regulation. Thus, our study showed the complex nature of SSP signaling and shed light on the regulation of SSPs in different plant lineages during infection.

The molecular beacon (MB) strategy finds diverse applications in bioimaging, biosensing and disorders diagnostics. Classic MBs are stem-loop oligonucleotides modified with a reporter and a quencher at the 5'- and 3'- ends. Hybridization with a specific target leads to spatial separation of the quencher and the reporter with subsequent changes in spectral signal, for example, an increase in reporter fluorescence emission. However, hydrophobic interactions between a conventional bulky quencher and the reporter might influence hybridization properties and limit stem length in MB design. In this work, we studied the synthetic nucleobase analog tCoAzo mimicking cytosine, capable of quenching reporter (FAM) fluorescence in folded MBs while minimally affecting their tharmal stability compared to MBs with classic FAM/BHQ1 labels. In addition, we conducted a preliminary assessment of the applicability of FAM/tCoAzo-modified MBs for SERS-based techniques using short single-stranded and long double-stranded DNA fragments from Fusarium avenaceum elongation factor 1a DNA as matrices. SERS intensity was decreased in the presence of matrix DNA compared to the scrambled sequence; and the MB modified with three tCoAzo residues provided a concentration-dependent signal. The results indicate that tCoAzo is a promising tool for fine tuning MB properties for fluorescent- and SERS-based diagnostic applications.

A rapid and relatively cost-effective method was developed to isolate and purify RNA from mycelia of two plant-pathogenic fungal species of the genus Fusarium, F. graminearum and F. coffeatum, with different morphological and biochemical properties. The method utilizes a guanidine hydrochloride-based buffer and spin columns from a commercial plasmid DNA extraction kit and can be applied to both mycelia grown on nutrient agar media and liquid fungal cultures. The RNA yield with the method was 4‒14 µg/100 mg of mycelium dry weight, and the RNA integrity number (RIN) values were up to 8.4. Method optimization showed that preliminary freeze drying is advisable to perform in the case of liquid cultures and that an RNase inhibitor should be used in the case of late culture growth stages.

PD1/PD-L1 antibody therapy is used in the clinic (atezolizumab, avelumab, durvalumab), but its effect is ambiguous, both in patients and in mouse models. The purpose of this work was to analyze the binding of commercial and laboratory-obtained monoclonal antibodies to PD-L1. Previously, we obtained monoclonal antibodies (clone B12) to the extracellular fragment of murine PD-L1 expressed in E. Coli (exPD-L1). The primary screening of monoclonal antibodies by flow cytometry showed a low B12 binding compared to commercial antibodies. Cytokine-stimulated 3D cultures, permeabilized cells, and Western blotting were used to elucidate the causes of low binding on living cells. The permeabilization of cells B16/F10 (mouse melanoma), EL-4 (mouse lymphoma), and COLO357 (human pancreatic cancer) showed the binding of both mouse and human B12 PD-L1 antibodies. With the help of Western blotting, these data were confirmed. However, commercial antibodies (BioLegend, clone 10F.9G2) in this method did not bind to the mouse recombinant exPD-L1 protein, but bound to cell lysates. Cell cultivation on an anti-adhesive polyHEMA substrate led to the binding of B12 antibodies to the surface of cells under 3D cultivation conditions. The stimulation of EL-4 and B16/F10 cells in 3D cultures and subsequent incubation with Cy3-labeled B12 antibodies led to the appearance of pronounced fluorescence in the perinuclear region of cells. Thus, the obtained monoclonal antibodies to mouse exPD-L1 are cross-reactive with human PD-L1 protein; PD-L1 protein translocates to the cell membrane when cultured under 3D conditions into the nucleus and, when stimulated by cytokines, its biosynthesis is activated. The difference in the binding of antibodies B12 and 10F.9G2 may indicate a difference in the conformation of PD-L1 translocated to the membrane. The traffic of intracellular PD-L1 into the nucleus upon activation may indicate the launch of de novo protein synthesis with an altered conformation.

Background/Objectives: Targeted delivery of chemotherapeutic agents is a well-established approach to cancer therapy. Antibody–drug conjugates (ADCs) typically carry toxic payloads attached to a tumor-associated antigen-targeting IgG antibody via an enzyme-cleavable linker that releases the drug inside the cell. Aptamers are a promising alternative to antibodies in terms of antigen targeting; however, their polynucleotide nature and smaller size result in a completely different PK/PD profile compared to an IgG. This may prove advantageous: owing to their lower molecular weight, aptamer-drug conjugates may achieve better penetration of solid tumors compared to ADCs. Methods: On the way to therapeutic aptamer–drug conjugates, we aimed to develop a versatile and modular approach for the assembly of aptamer–enzymatically cleavable payload conjugates of various drug–aptamer ratios. We chose the epidermal growth factor receptor (EGFR), a transmembrane protein often overexpressed in brain tumors, as the target antigen. We used the 46 mer EGFR-targeting DNA sequence GR-20, monomethylauristatin E (MMAE) on the cathepsin-cleavable ValCit-p-aminobenzylcarbamate linker as the payload, and pentaerythritol-based tetraazide as the branching point for the straightforward synthesis of aptamer–drug conjugates by means of a stepwise Cu-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. Results: Branched aptamer conjugates of 1:3, 2:2, and 3:1 stoichiometry were synthesized and showed higher cytotoxic activity compared to a 1:1 conjugate, particularly on several glioma cell lines. Conclusions: This approach is convenient and potentially applicable to any aptamer sequence, as well as other payloads and cleavable linkers, thus paving the way for future development of aptamer–drug therapeutics by easily providing a range of branched conjugates for in vitro and in vivo testing.

The problem of detecting tiny quantities of analytes by immunochemical methods has been tried for a long time. One approach is to use nucleic acid amplification methods to amplify the signal from a single antigen-antibody interaction. An amplification method suitable for microarrays is the rolling circle amplification reaction. The principle of the method is usage of a conjugate of a detecting antibody with a primer and subsequent isothermal amplification. The generation of a huge single-stranded reaction product starts after adding the necessary components for amplification reaction: circular oligonucleotides, which serves as a template for amplification and phage phi29 polymerase with the other components. This reaction product consists of a lot of repeats of a nucleotide sequence, that is complementary to the circular template. The fluorescent DNA probe can hybridize to each repeat on the product molecule, resulting in a significantly higher level of fluorescence than with fluorescently labeled antibody or streptavidin development systems. In addition, the reaction product remains immobilized on the surface, allowing usage of this approach for the detection of antigen-antibody interactions in other solid-phase analysis systems, such as microarrays. A common problem with such approaches is the nonspecific sorption of components of the immunochemical reaction or amplification reaction, leading to a high background. It is obvious that no matter how highly sensitive the analysis is in theory, a high background will reduce the entire potential of the method to nothing. Herein, we have developed a method that makes it possible to detect small amounts of antibodies to glycans in blood serum and in swabs from tumor cells in a microarray format using a model of blood group antigens. It was possible to obtain a 30 to 70-fold increase of fluorescence level from a specific interaction compared to the use of fluorescently labeled streptavidin. The method we are developing is promising, as it allows us to significantly increase the signal from the specific antigen/antibody interaction in the glycochip format, which will make it possible to detect antibodies to glycans in samples with a very low concentrations of antibodies, for example, in washes from tumor cells.

Objective: A number of molecules expressed on mammalian cells are involved in the formation of auto-tolerance. These primarily include CTLA-4/B7 and PD1-PD-L1 signaling pathways. Blockers of these signaling pathways, called checkpoint inhibitors (ICTs) of immunity, are used in the clinic for the treatment of various forms of cancer. Antibodies to CTLA-4 cause systemic toxicity and are approved only for the treatment of melanoma. Antibodies against PD1 or PD-L1 have been successfully used for the treatment of various forms of cancer and are characterized by low toxicity. However, the response to therapy using ICT does not exceed 25–30

Generally, lncPEPs (peptides encoded by long non-coding RNAs) have been identified in many plant species of several families and in some animal species. Importantly, molecular mechanisms of the miPEPs (peptides encoded by primary microRNAs, pri-miRNAs) are often poorly understood in different flowering plants. Requirement for the additional studies in these directions is highlighted by alternative findings concerning positive regulation of pri-miRNA/miRNA expression by synthetic miPEPs in plants. Further extensive studies are also needed to understand the full set of their roles in eukaryotic organisms. This review mainly aims to consider the available data on the regulatory functions of the synthetic miPEPs. Studies of chemically synthesized miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed. Brief description of the studies to identify lncORFs (open reading frames of long non-coding RNAs) and the encoded protein products is also provided.

Detecting small amounts of analytes, especially antibodies, presents a significant challenge in high-throughput methods. Fortunately, nucleic acid amplification techniques provide a promising solution. We have successfully developed and tested an innovative technology for detecting anti-glycan antibodies, utilizing a printed glycan array combined with a rolling circle amplification reaction based on the ABO blood group antibody model. This breakthrough has dramatically enhanced the sensitivity of our immunoassay, improving it by over an order of magnitude and allowing us to detect concentrations as low as 1 ng/ml. This advancement opens new approaches for research and clinical applications, making previously undetectable analytes accessible for study. ### Competing Interest Statement The authors have declared no competing interest.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

A universal approach to the construction of antibody-drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug-antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

Oligomerization of antibody fragments via modification with polyethylene glycol (pegylation) may alter their function and properties, leading to a multivalent interaction of the resulting constructs with the target antigen. In a recent study, we generated pegylated monomers and multimers of scFv fragments of GD2-specific antibodies using maleimide–thiol chemistry. Multimerization enhanced the antigen-binding properties and demonstrated a more efficient tumor uptake in a syngeneic GD2-positive mouse cancer model compared to monomeric antibody fragments, thereby providing a rationale for improving the therapeutic characteristics of GD2-specific antibody fragments. In this work, we obtained pegylated conjugates of scFv fragments of GD2-specific antibodies with maytansinoids DM1 or DM4 using tetravalent PEG-maleimide (PEG4). The protein products from the two-stage thiol–maleimide reaction resolved by gel electrophoresis indicated that pegylated scFv fragments constituted the predominant part of the protein bands, and most of the scFv formed pegylated monomers and dimers. The conjugates retained the ability to bind ganglioside GD2 comparable to that of the parental scFv fragment and to specifically interact with GD2-positive cells. Both induced significant inhibitory effects in the GD2-positive B78-D14 cell line, in contrast to the GD2-negative B16 cell line. The decrease in the B78-D14 cell viability when treated with scFv-PEG4-DM4 was more prominent than that for scFv-PEG4-DM1, and was characterized by a twofold lower half-maximal inhibitory concentration (IC50). Unlike the parental scFv fragment, the product of scFv and PEG4 conjugation (scFv–PEG4), consisting predominantly of pegylated scFv multimers and monomers, induced direct cell death in the GD2-positive B78-D14 cells. However, the potency of scFv–PEG4 was low in the selected concentration range, thus demonstrating that the cytotoxic effect of DM1 and DM4 within the antibody fragment–drug conjugates was primary. The suggested approach may contribute to development of novel configurations of antibody fragment–drug conjugates for cancer treatment.

Colletotrichum coccodes causes dangerous potato and tomato diseases, known as anthracnose and black spot. Morphologically, they are often difficult to distinguish from diseases caused by other microorganisms. On green tomato fruits, the disease may be asymptomatic; it appears only on ripe red fruits. For quick and accurate diagnosis and identification of the pathogen, a test system for real-time PCR is proposed. To develop a test system, the nucleotide sequence of the glycerol-3-phosphate dehydrogenase gene was determined in 45 different strains of C. coccodes . Based on the results obtained and analysis of similar sequences of other species available in the GenBank Database, C. coccodes species-specific primers and a probe were designed. To test the specificity of the created test system, PCR was done with DNA isolated from pure cultures of 15 different species of parasitic and saprotrophic fungi associated with tomato and potato plants ( Fusarium oxysporum , Fusarium verticillium , Phomopsis phaseoli , Alternaria alternatа , Helminthosporium solani , Colletotrichum coccodes , Phellinus ferrugineovelutinus , Stemphylium vesicarium , Helminthosporium solani , Phomopsis phaseoli , Neonectria radicicola , Rhizoctonia solani , Penicillium sp., Cladosporium fulvum , and Cladosporium cladosporioides ). The presence of Colletotrichum coccodes DNA was determined at a 20‒27 threshold cycle. The remaining types of fungi were determined after 40 cycles or were not detected at all. The test system makes it possible to confidently detect in the analyzed PCR-mixture concentrations of C. coccodes exceeding 0.01 ng/mm 3 . The test system was used to study the presence of C. coccodes in tomato leaves with symptoms of fungal diseases and potato tubers without external symptoms of disease. Leaves with fungal lesion symptoms were collected from two different fields in Krasnodar krai; tubers, from the fields in Kostroma, Moscow, Kaluga, and Nizhny Novgorod oblasts. A single tomato leaf containing C. coccodes DNA was found, while the presence of C. coccodes DNA tubers grown in Kostroma, Moscow, and Kaluga oblasts was detected in five samples.

Using a combination of solid-phase extraction, affinity chromatography, and analytical reverse-phase HPLC, a new linear peptide was isolated from dog-grass (Elytrigia repens) ears that does not contain cysteine residues. Identification of its primary structure by Edman automated degradation made it possible to reveal the presence of several polyglycine regions, each consisting of six to eight residues, between which short fragments consisting of polar amino-acid residues are localized. The C-terminal fragment of the molecule is a positively charged site enriched in arginine and histidine residues. The structural features of this peptide determine its functionality. Thus, checking the presence of antimicrobial properties in its recombinant analogue, obtained by heterologous expression in a prokaryotic system, made it possible to determine the MIC for the tested fungal cultures only at sufficiently high active concentrations (52–104 μM). However, this compound had regulatory properties: at a concentration of 25 μM, a reactivating effect was noted, which increased the level of survival of Saccharomyces cerevisiae to UV-irradiation. The data we obtained expand the understanding of the functional features of plant defense peptides of an unusual structural type.

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.

This study aimed to obtain a recombinant chimeric protein named trx-NsW2 via theheterologous expression of the multifunctional antimicrobial peptide nigellothionin from black cumin (Nigella sativa L.) seeds in the Escherichia coli system. The protein was purified using a combination of Ni-NTA affinity chromatography and reversed-phase HPLC. Based on the HPLC calibration, the total yield of the protein was calculated to be 650 mg/L of bacterial culture. The fungistatic activity of trx-NsW2 against the food-spoiling fungus Aspergillus niger was demonstrated as itinhibited the maturation of conidiawithout affecting conidial germination or fungal growth. In contrast to mature nigellothionin NsW2, the fusion protein showeda low level of cytotoxicity towards both normal and tumor cell lines at concentrationsof up to 100–200 µM. Interestingly, at lower concentrations, it even stimulated cytokinesis. These findings are of critical importance for applying chimeric antimicrobial proteins obtained via microbiological synthesis in applied science.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties–minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

Plant antimicrobial peptides from the α-hairpinins family (hairpin-like peptides) are known to possess a wide range of biological activities. However, less is known about the structural determinants of their antimicrobial activity. Here, we suggest that spatial structure as well as surface charge and hydrophobicity level contribute to the antimicrobial properties of α-hairpinin EcAMP1 from barnyard grass (Echinochloa cruss-galli) seeds. To examine the role of the peptide spatial structure, two truncated forms of EcAMP1 restricted by inner and outer cysteine pairs were synthesized. It was shown that both truncated forms of EcAMP1 lost their antibacterial activity. In addition, their antifungal activity became weaker. To review the contribution of surface charge and hydrophobicity, another two peptides were designed. One of them carried single amino acid substitution from tryptophan to alanine residue at the 20th position. The second one represented a truncated form of the native EcAMP1 lacking six C-terminal residues. But the α-helix was kept intact. It was shown that the antifungal activity of both modified peptides weakened. Thereby we can conclude that the secondary structural integrity, hydrophobic properties, and surface charge all play roles in the antimicrobial properties of α-hairpinins. In addition, the antibacterial activity of cereal α-hairpinins against Gram-positive bacteria was described for the first time. This study expands on the knowledge of structure–function interactions in antimicrobial α-hairpinins.

The article discusses new data obtained in the study of the functions of a conservative peptide of cabbage plants, which is encoded by the microRNA156a. Comparative analysis of the nucleotide sequences of the promoter regions of these genes allowed us to identify a highly conserved 42-residue block located before the starting point of pri-miR156a transcription at a distance of 210-260 base pairs. It was found that promoter fragments containing a highly con-served block have a significantly higher ability to bind miPEP156a in vitro. We carried out mutagenesis of a highly conserved promoter block in its central part, which includes a tetramer of TG dinucleotides. It has been shown that the introduction of mutations into the promoter tetramer of TG dinucleotides significantly reduces the affinity of the promoter DNA to miPEP156a. The miPEPs revealed in plants have been found only in dicotyledons. The question of how miPEPs are distributed in other plant taxa is very important for understanding the evo-lutionary origin of such micropeptides. As an initial approach, we searched for taxonomically conservative miPEPs in mosses, since microRNAs have been studied in a great detail in the case of Physcomitrium patens moss. For two genes in the region preceding the Ppt-pre-miR160a sequence, rather short open reading frames were found that encoded peptides having a clear similarity of amino acid sequences in the central region. Importantly, such highly con-served peptide block homologous to that encoded by Ppt-miPEP160a gene was detected in short proteins encoded in pri-miR160a in almost 20 Bryopsida mosses.

Abstract This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.