Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research.

Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.

Targeting Tumor Markers with Antisense Peptides: An Example of Human Prostate Specific Antigen.

The purpose of this paper was to outline the development of short peptide targeting of the human prostate specific antigen (hPSA), and to evaluate its effectiveness in staining PSA in human prostate cancer tissue. The targeting of the hPSA antigen by means of antisense peptide AVRDKVG was designed according to a three-step method involving: 1. The selection of the molecular target (hPSA epitope), 2. the modeling of an antisense peptide (paratope) based on the epitope sequence, and 3. the spectroscopic evaluation of sense-antisense peptide binding. We then modified standard hPSA immunohistochemical staining practice by using a biotinylated antisense peptide instead of the standard monoclonal antibody and compared the results of both procedures. Immunochemical testing on human tissue showed the applicability of the antisense peptide technology to human molecular targets. This methodology represents a new approach to deriving peptide ligands and potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

Hepatoprotective effects of Met-enkephalin on acetaminophen-induced liver lesions in male CBA mice.

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p>0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Level of primary DNA damage in the early stage of metabolic syndrome.

Metabolic syndrome (MetS) is a multi-component disease, characterised by abdominal obesity, hypertension, hyperglycaemia and dyslipidaemia. Since the number of MetS patients has significantly increased over the past two decades and because MetS may lead to development of cardiovascular diseases, diabetes type-2, and cancer, it has become important to extend the knowledge on the pathogenesis of MetS and to establish its possible early biomarkers. Studies on MetS and DNA damage are few and are inconclusive. The aim of this study was to elucidate the involvement of DNA damage in the development of MetS and to establish if DNA damage can serve as early biomarker of MetS. A total of 121 subjects participated in the study: 56 healthy controls and 65 MetS patients who were diagnosed with MetS for the first time. The amount of primary DNA damage in peripheral leukocytes of the subjects was assessed with three types of comet assay: the alkaline, the hOGG1-modified, and the neutral comet assay. In addition, the extent of oxidative DNA damage was monitored in urine by assessing 8-oxo-dG. The parameters of the three types of comet assay did not differ between the control and the MetS group. Interestingly, urinary 8-oxo-dG level in the control group was higher than in the MetS group. Our results imply that DNA damage is not involved in the early stage of MetS and, therefore, DNA damage cannot serve as an early marker of MetS.

Immune thrombocytopenia: serum cytokine levels in children and adults.

BACKGROUND: Immune thrombocytopenia (ITP) is an immune-mediated platelet disorder in which autoantibody-coated platelets are removed from the blood by monocytic phagocytes and there is impaired platelet production. There is a delicate balance of specific cytokine levels, which has an important role in the immune system and is known to be deregulated in autoimmune diseases. This study was designed to investigate the differences in Th cytokine levels between children and adults with newly diagnosed ITP and to compare these profiles to those found in healthy, age-matched controls. MATERIAL/METHODS: The concentration of IL-1alpha, IL-2, IL-3, IL-4, IL-6, IL-10, TNF-alpha, IFN-alpha, and IFN-alpha in serum specimens was analyzed by enzyme-linked immunosorbent assay. RESULTS: At the time of ITP diagnosis, children showed significantly lower serum levels of interleukin IL-2 and tumor necrosis factor TNF-alpha and higher serum level of IL-3 than healthy controls. Serum level of IL-4 in adult ITP patients was higher than those in control subjects. When compared with adults, children with ITP had lower serum level of IL-4, IL-6 and IFN-alpha, and higher level of IFN-alpha. CONCLUSIONS: Significant differences in serum cytokine levels between pediatric patients and healthy controls indicate that cytokine disturbances--especially changes in IL-2, IL-3 and TNF-alpha--might be involved in the pathogenesis of newly diagnosed ITP. TNF-alpha is the most informative variable for discrimination between healthy children and those with ITP.

Extracellular Matrix- and Integrin Adhesion Complexes-Related Genes in the Prognosis of Prostate Cancer Patients' Progression-Free Survival.

Prostate cancer is a heterogeneous disease, and one of the main obstacles in its management is the inability to foresee its course. Therefore, novel biomarkers are needed that will guide the treatment options. The extracellular matrix (ECM) is an important part of the tumor microenvironment that largely influences cell behavior. ECM components are ligands for integrin receptors which are involved in every step of tumor progression. An underlying characteristic of integrin activation and ligation is the formation of integrin adhesion complexes (IACs), intracellular structures that carry information conveyed by integrins. By using The Cancer Genome Atlas data, we show that the expression of ECM- and IACs-related genes is changed in prostate cancer. Moreover, machine learning methods revealed that they are a source of biomarkers for progression-free survival of patients that are stratified according to the Gleason score. Namely, low expression of FMOD and high expression of PTPN2 genes are associated with worse survival of patients with a Gleason score lower than 9. The FMOD gene encodes protein that may play a role in the assembly of the ECM and the PTPN2 gene product is a protein tyrosine phosphatase activated by integrins. Our results suggest potential biomarkers of prostate cancer progression.

How ambiguity codes specify molecular descriptors and information flow in Code Biology.

The article presents IUPAC ambiguity codes for incomplete nucleic acid specification, and their use in Code Biology. It is shown how to use this nomenclature in order to extract accurate information on different properties of the biological systems. We investigated the use of ambiguity codes, as mathematical and logical operators and truth table elements, for the encoding of amino acids by means of the Standard Genetic Code. It is explained how to use ambiguity codes and truth functions in order to obtain accurate information on different properties of the biological systems. Nucleotide ambiguity codes could be applied to: 1. encoding descriptive information of nucleotides, amino acids and proteins (e.g., of polarity, relative solvent accessibility, atom depth, etc.), and 2. system modelling ranging from standard bioinformatics tools to classic evolutionary models (i.e. from Miyazawa-Jernigan statistical potential to Kimura three-substitution-type model, respectively). It is shown that the algorithms based on IUPAC ambiguity codes, Boolean functions and truth table, Probabilistic Square of Opposition/Semiotic Square and Klein 4-groups-could be used for the bioinformatics analyses and Relational data modelling in natural science. Underlying mathematical, logical and semiotic concepts of interest are presented and addressed.

Prognostic Significance of Amino Acid Metabolism-Related Genes in Prostate Cancer Retrieved by Machine Learning.

Prostate cancer is among the leading cancers according to both incidence and mortality. Due to the high molecular, morphological and clinical heterogeneity, the course of prostate cancer ranges from slow growth that usually does not require immediate therapeutic intervention to aggressive and fatal disease that spreads quickly. However, currently available biomarkers cannot precisely predict the course of a disease, and novel strategies are needed to guide prostate cancer management. Amino acids serve numerous roles in cancers, among which are energy production, building block reservoirs, maintenance of redox homeostasis, epigenetic regulation, immune system modulation and resistance to therapy. In this article, by using The Cancer Genome Atlas (TCGA) data, we found that the expression of amino acid metabolism-related genes is highly aberrant in prostate cancer, which holds potential to be exploited in biomarker design or in treatment strategies. This change in expression is especially evident for catabolism genes and transporters from the solute carrier family. Furthermore, by using recursive partitioning, we confirmed that the Gleason score is strongly prognostic for progression-free survival. However, the expression of the genes SERINC3 (phosphatidylserine and sphingolipids generation) and CSAD (hypotaurine generation) can refine prognosis for high and low Gleason scores, respectively. Therefore, our results hold potential for novel prostate cancer progression biomarkers.

New algorithm for the analysis of nucleotide and amino acid evolutionary relationships based on Klein four-group.

Phylogenetics is the study of ancestral relationships among biological species. Such sequence analyses are often represented as phylogenetic trees. The branching pattern of each tree and its topology reflect the evolutionary relatedness between analyzed sequences. We present a Klein four-group algorithm (K4A) for the evolutionary analysis of nucleotide and amino acid sequences. Klein four-group set of operators consists of: identity e (U), and three elements-a = transition (C), b = transversion (G) and c = transition-transversion or complementarity (A). We generated Klein four-group based distance matrices of: 1. Cayley table (CK4), 2. Table rows (K4R), 3. Table columns (K4C), and 4. Euclidean 2D distance (K4E). The performance of the matrices was tested on a dataset of RecA proteins in bacteria, eukaryotes (Rad51 homolog) and archaea (RadA homolog). RecA and its functional homologs are found in all species, and are essential for the repair and maintenance of DNA. Consequently, they represent a good model for the study of evolutionary relationship of protein and nucleotide sequences. The ancestral relationship between the sequences was correctly classified by all K4A matrices concerning general topology. All distance matrices exhibited small variations among species, and overall results of tree classification were in agreement with the general patterns obtained by standard BLOSUM and PAM substitution matrices. During the evolution of a code there is a phase of optimization of system rules, the ambiguity of a code is eliminated, and the system starts producing specific components. Klein four-group algorithm is consistent with the concept of ambiguity reduction. It also enables the use of different genetic code table variants optimized for particular transitions in evolution based on biological specificity.

The effects of simvastatin and fenofibrate on malondialdehyde and reduced glutathione concentrations in the plasma, liver, and brain of normolipidaemic and hyperlipidaemic rats.

The objective of study was to investigate the effects of different doses of simvastatin and fenofibrate on malondialdehyde (MDA) and reduced glutathione (GSH) in the plasma, liver, and brain tissue of male normolipidaemic and hyperlipidaemic rats. Normolipidaemic (Wistar) rats were receiving 10 or 50 mg/kg a day of simvastatin or 30 or 50 mg/kg a day of fenofibrate. Hyperlipidaemic (Zucker) rats were receiving 50 mg/kg/day of simvastatin or 30 mg/kg/day of fenofibrate. Control normolipidaemic and hyperlipidaemic rats were receiving saline. Simvastatin, fenofibrate, and saline were administered by gavage for three weeks. In normolipidaemic rats simvastatin and fenofibrate showed similar and dose-independent effects on plasma and brain MDA and GSH concentrations. Generally, plasma and brain MDA decreased, while brain GSH concentration increased. In hyperlipidaemic rats simvastatin did not affect plasma and brain MDA and GSH concentrations but significantly decreased liver GSH. Fenofibrate decreased plasma and liver MDA but increased brain MDA. In both rat strains fenofibrate significantly decreased liver GSH concentrations, most likely because fenofibrate metabolites bind to GSH. Our findings suggest that simvastatin acts as an antioxidant only in normolipidaemic rats, whereas fenofibrate acts as an antioxidant in both rat strains.

Idiopathic nephrotic syndrome in children: review of 282 Croatian cases.

Recent data suggests increased incidence of focal segmental glomerulosclerosis (FSGS) among children with idiopathic nephrotic syndrome (INS). To determine the causes and possible longitudinal changes in the etiology of INS, 282 Croatian children diagnosed with INS between 1990 and 2009 were evaluated. In total, 122 children were assessed as having minimal change nephrotic syndrome (MCNS) based on their initial presentation, laboratory findings and clinical course. Kidney biopsy was performed in the remaining 160 children. MCNS was present in 18.1% of all biopsies performed. Total incidence of MCNS (assessed + biopsy proven) was only 53.5%. In contrast, FSGS was found in 40.6% of all biopsies and accounted for 23.1% of all cases. Mesangial proliferative glomerulonephritis (MesPGN) was the third most common diagnosis, present in 26.9% of the biopsies, and accounted for 15.2% of all cases. There were no significant longitudinal differences in the incidence of different causes of INS. The overall response to steroids at presentation was 71.6%. A higher proportion of initial steroid responders among children with FSGS (43.1%) and MesPGN (67.4%) than previously reported was noted. A longitudinal tendency of increasing steroid resistance in FSGS and MesPGN groups was observed.

Cytoprotective effects of ß-melanocortin in the rat gastrointestinal tract.

Recently discovered anti-inflammatory and immunomodulatory properties of melanocortin peptides led to the conclusion that they might serve as new anti-inflammatory therapeutics. The purpose of this work was to examine the effectiveness of ß-melanocortin (ß-MSH) in two experimental models: ethanol-induced gastric lesions and TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced colitis in male Wistar rats. Three progressive doses of ß-MSH were used: 0.125, 0.250 and 0.500 mg/kg. Our results suggest that ß-MSH acts as a protective substance in the gastric lesions model, which can be seen as a statistically significant reduction of hemorrhagic lesions at all three doses, compared to the control group. The most efficient dose was 0.250 mg/kg. Statistically significant reduction in mucosal surface affected by necrosis and the reduction of overall degree of inflammation in the colitis model indicates an anti-inflammatory effect of ß-MSH at a dose of 0.250 mg/kg. The results justify further research on ß-MSH peptide and its derivates in the inflammatory gastrointestinal diseases, and point out the possibility of using ß-MSH in studies of digestive system pharmacology.

Effects of alpha-MSH on corneal epithelial lesions in rats.

We investigated the influence of alpha-melanocyte stimulating hormone (alpha-MSH) on the healing of corneal epithelial lesions in male Wistar rats. The lesions were induced by abrading corneal epithelium with a surgical lancet under a biomicroscope. Before the abrasion corneal epithelium was softened with 40% ethanol. alpha-MSH drops in a concentration of 1 x 10(-4) mg/mL significantly promoted epithelial healing, when compared to the control group treated with 0.9% NaCl. Significant reduction of ocular irritation was also observed. Topical alpha-MSH medication could promote the healing of corneal wounds and reduce local inflammatory response without the major side effects of the corticosteroid therapy.

Ochratoxin A potentiates citrinin accumulation in kidney and liver of rats.

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3.

The role of independent test set in modeling of protein folding kinetics.

The testing of a bioinformatics algorithm on the training set is not the best indicator of its future performance because of the misleadingly optimistic results. The optimal method of testing is the calculation of error rate on an independent dataset (test set). We have tested the validity of the FOLD-RATE method for the prediction of protein folding rate constants [ln(k ( f ))] using sequences, structural class information and experimentally verified folding rate constants of the Protein Folding Database (PFD). PFD is a publicly accessible repository of thermodynamic and kinetic data of interest for the researchers of different profiles, standardized by the International Foldeomics Consortium. Our results show that when the standardized PFD dataset is used to test a protein fold rate prediction method, the estimation of validity may differ significantly.

Interaction of α-melanocortin and its pentapeptide antisense LVKAT: effects on hepatoprotection in male CBA mice.

The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of continuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3'→5' direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo.

Relational model of the standard genetic code.

The genetic code is a set of rules that establishes mapping between triplets in messenger RNA and amino acids in proteins. The most common way to display these rules is the Standard Genetic Code (SGC) table. This paper takes an alternative approach, based on the relational data model by Edgar F. Codd (Commun. ACM, 13:377-387, 1970). The relational model (RM) proposes a distributed storage of data into a collection of tables (called relations), that can be connected by shared communality. Basic elements of the table are rows (called records or tuples), and columns (called fields or attributes). The SGC table, according to the relational data model, represents the so called unnormalized form of a table. Using normalization rules it is possible to subdivide the SGC table into four tables. The rows and columns of single tables are defined by the first and second base and individual tables by the third codon base. The result of this model is an approach to managing genetic code data, represented in terms of tuples and grouped into relations, with table structure and language consistent with first-order (predicate) logic. The RM explains that the final step in the development of the SGC was the adoption of coding function by the third base, which makes an informational/functional unit with the first base, despite the different physical location in a triplet. This enabled the synthesis of specific proteins without ambiguity, in accordance with the concept of ambiguity reduction and five phases of the general model on the origin of biological codes by Marcello Barbieri (BioSystems 181:11-19, 2019).

The influence of alpha-, beta-, and gamma-melanocyte stimulating hormone on acetaminophen induced liver lesions in male CBA mice.

Research over the past decade has indicated that melanocortin peptides are potent inhibitors of inflammation and a promising source of new anti-inflammatory and cytoprotective therapies. The purpose of the present paper is to compare protective effects of alpha-, beta-, and gamma-melanocyte stimulating hormone on acetaminophen induced liver lesions in male CBA mice. Acetaminophen was applied intragastrically in a dose of 150 mg/kg, and tested substances were applied intraperitoneally 1 hour before acetaminophen. Mice were sacrificed after 24 hours and intensity of liver injury was estimated by measurement of plasma transaminase activity (AST and ALT) and histopathological grading of lesions. It was found that alpha-, beta-, and gamma-MSH decrease intensity of lesions by both criteria in a dose-dependent manner.

Cystitis cystica and recurrent urinary tract infections in children.

The pathogenesis of recurrent urinary tract infections (UTIs) in preschool children with anatomically correct urinary tract (UT) is rather obscure. In girls, the bladder wall changes of cystitis cystica (CC) may be per se responsible for UTIs recurrence. During the 20-year period, 127 preschool children (125 girls; median age: 6.1 years) with CC, in whom UT anomalies were excluded, were diagnosed. The mean duration of UTIs symptoms prior to diagnosis was 3.31 +/- 2.51 years. Cystoscopical findings were labelled as mild, moderate and severe in 22.8%, 39.4% and 37.8% of patients, respectively. Following the confirmation of CC, long-term chemoprophylaxis with sulfamethoxazole-trimethoprim/nitrofurantoin was administered. A one year UTI-free period after chemoprophylaxis discontinuation was defined as therapeutic success. With 2.5 years median duration of regular chemoprophylaxis this goal was achieved in 58 children mainly with mild/ moderate CC. Thirty children from "improved/unchanged" group taking regular prophylaxis had significant reduction of UTIs ("improved"). Only 12 children belonging to the same group taking regular prophylaxis and all children with irregular prophylaxis had approximately the same number of UTIs as before treatment ("unchanged"). The "improved/unchanged" outcomes were predominantly found in children with severe form of CC. Although urodynamic disturbances detected in more than 50% of patients in whom urodynamics was performed were not found influential on the disease outcome, they could be responsible for its development. The results of our study suggest that regular and long-lasting chemoprophylaxis remains a basis for successful treatment for majority of patients with CC, even those with severe forms. If not treated properly with chemoprophylactic agents and without fair compliance in taking drugs, the disease is prone to recurrent UTIs.