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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.

The quantity and quality of preoperative material in colorectal cancer is often limiting factor in determination of risk factors and therapy planning. The most important negative prognostic factors are intravascular and perineural invasion, as well as tumor budding. Usually, the only parameter available in preoperative biopsy is tumor budding. However, the growing body of evidence suggests that cancer differentiation based on the poorly differentiated clusters has better prognostic value. The limiting factor in applying of these new parameters is reproducible, simple, cheap and fast method of their determination. In this paper we investigated the prognostic value of lacunarity, determined in preoperative biopsy. Lacunarity is a measure of spatial heterogeneity (inhomogeneity) in an image. It quantifies how objects fill the space, and enables analysis of gaps distribution, homogeneity of gaps, and presence of structures. It was shown that lacunarity and the total number of buds could be combined in a model which clearly divides colorectal cancer patients in low, medium and high risk subgroups. The paper also points out that the quantitative numerical methods are superior to semiquantitative methods, and that individual methods should be combined using algorithms to obtain a more accurate prediction. Because the study described is designed as a pilot study, verification is needed on a larger sample of patients from independent researchers.

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).

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.

We present the data concerning the clustering of sense and antisense amino acid pairs into polar, nonpolar and neutral groups, as measured using hydrophobicity parameter—logarithmic equilibrium constants (Log10 Kw>c)—at 25 °C and 100 °C (Wolfenden et al., 2015). The Log10 Kw>c, values, of the complementary amino acid pairs are strongly correlated to the central (2nd) purine base of the mRNA codon and the complementary pyrimidine base of the tRNA anticodon. Clustering of amino acids is temperature independent with regard to the direction of translation (3′ → 5′ or 5′ → 3′). The Log10 Kw>c discriminate between artificial Hecht α- and β-protein datasets at 25 °C and 100 °C. Interpretation of this data may be found in the research article entitled “Determining amino acid scores of the genetic code table: complementarity, structure, function and evolution” (Stambuk and Konjevoda, 2020).

Nutritional stress disturbs the cellular redox-status, which is characterized by the increased generation of reactive oxygen species (ROS). The NRF2-NQO1 axis represents a protective mechanism against ROS. Its strength is cell type-specific. FaDu, Cal 27 and Detroit 562 cells differ with respect to basal NQO1 activity. These cells were grown for 48 hours in nutritional conditions (NC): (a) Low glucose&ndash, NC2, (b) no glucose, no glutamine&ndash, NC3, (c) no glucose with glutamine&ndash, NC4. After determining the viability, proliferation and ROS generation, NC2 and NC3 were chosen for further exploration. These conditions were also applied to IMR-90 fibroblasts. The transcripts/transcript variants of NRF2 and NQO1 were quantified and transcript variants were characterized. The proteins (NRF2, NQO1 and TP53) were analyzed by a western blot in both cellular fractions. Under NC2, the NRF2-NQO1 axis did not appear activated in the cancer cell lines. Under NC3, the NRF2-NQO1axis appeared slightly activated in Detroit 562. There are opposite trends with respect to TP53 nuclear signal when comparing Cal 27 and Detroit 562 to FaDu, under NC2 and NC3. The strong activation of the NRF2-NQO1 axis in IMR-90 resulted in an increased expression of catalytically deficient NQO1, due to NQO1*2/*2 polymorphism (rs1800566). The presented results call for a comprehensive exploration of the stress response in complex biological systems.

The Standard Genetic Code (SGC) table was investigated with respect to the three- dimensional codon arrangement, and all possible 24 hierarchical base partitions (4! = 24). This was done by determining the amino acid scores for each codon hierarchy in relation to the 1st horizontal, 2nd vertical and 3rd horizontal sub-tables. Marked differences were observed for the hydrophobicity and lipophilicity parameters encoded by the second base of the SGC table. The nucleotide hierarchy U < C < G < A and its complement A < G < C < U at the second base correlated best with the amino acid hydrophobicity and polarity. By contrast, the hierarchy C < G < U < A and its backwards transcript A < U < G < C at the second base were associated with the amino acid parameters of lipophilicity and accessible surface area. No association was observed between 24 base hierarchies of the codons at the 1st and 3rd positions with respect to the hydropathy, polarity, lipophilicity and accessible surface area. The results imply that the second base possesses the majority of information content with respect to the physicochemical properties observed. It is shown that amino acid information obtained by determining the scores of the bases and codon weightings in digital form coincides with physicochemical properties, and the temperature range between 25 °C and 100 °C does not affect the hydrophobicity, the related prediction of α- and β-protein structure, codon scores, or the complementarity code for sense and antisense peptide interactions. The amino acid scores determined for the SGC table enable the construction of rules and algorithms for the analysis of the structure, function and evolution of proteins. It has been demonstrated that IUPAC-based encoding of nucleobase and amino acid sequences could be used for the representation of the bases with the Semiotic (Greimas) Square and probabilistic square of opposition. It is concluded that the structural, functional and evolutionary patterns of the protein sequences may be modeled using codon based amino acid information, instead of using the information based on amino acid physicochemical properties only.

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&ndash, 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.

The study objective was to test the hypothesis that simvastatin and fenofibrate should cause an increase in butyrylcholinesterase (BuChE) activity not only in the plasma and liver but also in the brain of normolipidemic and hyperlipidemic rats. Catalytic enzyme activity was measured using acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) as substrates. Normolipidemic and hyperlipidemic rats were divided in four groups receiving 50 mg/kg of simvastatin a day or 30 mg/kg of fenofibrate a day for three weeks and three control groups receiving saline. Simvastatin and fenofibrate caused an increase in brain BuChE activity in both normo- and hyperlipidemic rats regardless of the substrate. The increase with BTCh as substrate was significant and practically the same in normolipidemic and hyperlipidemic rats after simvastatin treatment (14–17% vs controls). Simvastatin and fenofibrate also increased liver and plasma BuChE activity in both normolipidemic and hyperlipidemic rats regardless of the substrate. In most cases the increase was significant. Considering the important role of BuChE in cholinergic transmission as well as its pharmacological function, it is necessary to continue investigations of the effects of lipid-lowering drugs on BuChE activity., Cilj ispitivanja bio je u normolipidemičnih i hiperlipidemičnih štakora potvrditi pretpostavku da simvastatin (SIMV) I fenofibrat (FENO) ne uzrokuju samo povećanje aktivnosti butirilkolinesteraze (BuChE) u plazmi i jetri nego i povećavaju aktivnosti BuChE u mozgu. Katalitička aktivnost enzima mjerena je upotrebom acetiltiokolina (ATCh) i butiriltiokolina (BTCh) kao supstrata. Normolipidemični i hiperlipidemični štakori raspoređeni su u eksperimentalne skupine, koje su tri tjedna primale SIMV 50 mg/kg na dan ili FENO 30 mg/kg na dan, dok su kontrolne skupine primale fiziološku otopinu. I SIMV i FENO uglavnom su izazvali povećanje aktivnosti BuChE u mozgu obaju sojeva štakora bez obzira na korišteni supstrat. Aktivnosti BuChE mjerene BTCh kao supstratom bile su značajno veće u odnosu na kontrolne vrijednosti u mozgu normolipidemičih štakora nakon primjene SIMV te u mozgu hiperlipidemičnih štakora nakon primjene obaju agensa (14‒17%, p

Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder characterized by skin hyperextensibility, abnormal wound healing, and joint hypermobility with prevalence 1:20 000. Its incidence is probably underestimated due to unknown number of subjects having mild symptoms who may have never been diagnosed through entire life time. Classical EDS is characterized by pathogenic variants of genes encoding type V collagen. The biological effects and health risks of patients with EDS exposure to low doses of ionizing radiation is poorly understood. The aim of this study was to investigate biological effect of low doses of ionizing radiation in children with EDS. Background values of chromosome aberrations in children suffering from classical EDS were determined and compared with control subjects. The in vitro experiment was performed by γ-irradiation of blood lymphocytes from EDS patients and healthy subjects at low doses (0.1, 0.2 and 0.3 Gy). Results show a significant increase level of spontaneous and radiation-induced chromosomal aberrations in children suffering from EDS in comparison with the control subjects (p < 0.05). In conclusion, children with EDS express higher background chromosome aberration frequency and increased radiosensitivity. These findings suggest specific susceptibility of EDS patients and importance of future investigation on risks of diagnostics and therapy which include radiation and genotoxic agents.

Background: Breslow tumor thickness and mitotic rate are standardly used for risk stratification of patients with malignant melanoma. However, their prognostic value is relatively limited and a need for improved prognostication has been advocated. We aimed to screen the tumor tissue proteome in a search for potentially useful prognostic factors in early-stage cutaneous head and neck melanoma. ----- Methodology and findings: Proteomic profiles of archival formalin-fixed tissue samples of 31 patients (age 23-90 years) with early-stage head and neck cutaneous malignant melanoma (American Joint Committee on Cancer, AJCC, stage I/II) were determined and expression intensities were compared to those of melanocytic nevi, yielding ratios used in data analysis. Medical charts were retrospectively reviewed to determine time elapsed since diagnosis to disease-specific death or censoring. In a multivariate recursive partitioning analysis (as per AJCC guidelines), higher expression levels of heterogeneous nuclear ribonucleoprotein M (hnRNP M) [n = 18, HR = 1.94 vs. the entire cohort; HR = 5.95 (95%CI 2.43-14.5) for "high" vs. "low" (n = 13)] and of heat shock protein 90 alpha (HSP 90α) [n = 17, HR = 2.09 vs. the entire cohort; HR = 4.59 (95%CI 1.87-11.2) for "high" vs. "low" (n = 14)] were each independently strongly associated with higher mortality (accounting for clinical and standard pathohistological features). Higher Breslow thickness and mitotic rate were associated with higher mortality only when proteomic data were disregarded. ----- Conclusions and significance: Data suggest that tumor tissue expression of hnRNP M and/or of HSP 90α deserve further investigation and clinical validation as potential novel risk stratification aids in patients with stage I-II cutaneous head and neck malignant melanoma.

Aims To determine whether the brain disturbances develop in late-onset intrauterine growth restriction (IUGR) before blood flow redistribution towards the fetal brain (detected by Doppler measurements in the middle cerebral artery and umbilical artery). Further, to evaluate predictive values of Doppler arterial indices and umbilical cord blood gases and pH for early functional and/or morphological brain disturbances in late-onset IUGR. Study design This cohort study included 60 singleton term pregnancies with placental insufficiency caused late-onset IUGR (IUGR occurring after 34 gestational weeks). Umbilical artery resistance index (URI), middle cerebral artery resistance index (CRI), and cerebroumbilical (C/U) ratio (CRI/URI) were monitored once weekly. Umbilical blood cord samples (arterial and venous) were collected for the analysis of pO 2, pCO 2 and pH. Morphological neurological outcome was evaluated by cranial ultrasound (cUS), whereas functional neurological outcome by Amiel-Tison Neurological Assessment at Term (ATNAT). Results 50 fetuses had C/U ratio>1, and 10 had C/U ratio≤1; among these 10 fetuses, 9 had abnormal neonatal cUS findings and all 10 had non-optimal ATNAT. However, the total number of abnormal neurological findings was much higher. 32 neonates had abnormal cUS (53.37%), and 42 (70.00%) had non-optimal ATNAT. Furthermore, Doppler indices had higher predictive validity for early brain disturbances than umbilical cord blood gases and pH. C/U ratio had the highest predictive validity with threshold for adverse neurological outcome at value 1.13 (ROC analysis), i.e., 1.18 ( party machine learning algorithm). Conclusion Adverse neurological outcome at average values of C/U ratios>1 confirmed that early functional and/or structural brain disturbances in late-onset IUGR develop even before activation of fetal cardiovascular compensatory mechanisms, i.e., before Doppler signs of blood flow redistribution between the fetal brain and the placenta.

U radu je ispitivan utjecaj pet eteričnih ulja (anisa, paprene metvice, bosiljka, ružmarina i lavande prave) i njihovih dviju najzastupljenijih komponenti na porast micelija Colletotrichum coccodes, ekonomski značajne fitopatogene gljive i uspoređen s fungicidima. Ispitivanja su provedena u uvjetima in vitro u osam volumena na PDA podlozi u četiri ponavljanja. Porast micelija mjeren je osmi i petnaesti dan nakon nacjepljivanja micelija. Utvrđeno je da neka eterična ulja i komponente, primijenjeni u određenome volumenu, imaju signifikantan antifugalni učinak, u pojedinim primjerima usporediv s fungicidima. Najbolje djelovanje i najniži IC50 imala su eterična ulja anisa i paprene metvice te komponenta anetol. Petnaesti dan nakon inokulacije micelija eterična ulja imala su značajno bolje antifungalno djelovanje od svoje druge komponente po zastupljenosti., The effect of five essential oils (anise, peppermint, basil, rosemary and true lavander) and their two most common components on the mycelial growth in Colletotrichum coccodes, economically important phytopathogic fungi and compared with fungicides have been investigated in the study. Tests were conducted in vitro conditions in eight volumes on a PDA substrate in four replicates. The increase in mycelium was measured on the eighth and fifteenth day after the mycelium inoculation. It was found out that some essential oils and components applied at a given volume have a significant antifungal effect, in some examples comparable to fungicides. Anise and peppermint essential oils as well as the anethole component had the best activity and the lowest IC50. Fifteen days after the inoculation of the mycelium, the essential oils had a significantly better antifungal activity compared to their second most represented component.

Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

Aim: The association of the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with idiopathic nephrotic syndrome (INS) is controversial. Only scarce information on European populations is available. The aim of the study was to investigate the distribution of the ACE gene I/D polymorphism and its impact on INS in children from Croatia. Materials and Methods: Ninety-five children with INS were investigated: 30 with minimal change disease (MCD), 35 with mesangial proliferative glomerulonephritis (MesPGN) and 30 with focal segmental glomerulosclerosis (FSGS). The control group consisted of 73 healthy adults. ACE gene was analyzed using the PCR method. The results were correlated with clinical features, renal morphology and response to immunosuppresive therapy. Results: There was no correlation of ACE genotype with gender, age of the disease onset, level of proteinuria, presence of hematuria or hypertension, and GFR at onset of the disease. No statistically significant differences in ACE genotype or allele frequencies between the controls and whole group of patients, MCD group, MesPGN group, FSGS group, steroid sensitive (SS) patients, steroid resistant (SR) patients, as well as each other, were found, although DD genotype tended to be more frequent in FSGS patients, SR patients, and frequent relapsers. Among 11 children treated with cyclophosphamide the D allele was significantly higher among non-responders (p = 0.003). Conclusion: DD genotype is not a genetic risk factor for acquiring INS nor significant phenotype modifier regarding to clinical and pathohistological picture and response to steroids in Croatian children. The potential application of ACE genotyping in predicting cyclophosphamide response deserves further investigation.

A multimycotoxin analysis approach in grains results in frequent simultaneous findings of nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CTN). The mechanism of CTN and OTA toxicities in biological systems is not fully understood but it is known that oxidative stress is involved. In this study, oxidative damage of DNA, lipids, and the concentration of glutathione (GSH), as well as possible antioxidative effects of resveratrol (RSV) were studied in vivo. Male adult Wistar rats were treated orally with OTA (0.125 and 0.250 mg kg−1 b.w.), RSV (20 mg kg−1 b.w.) for 21 days, CTN (20 mg kg−1 b.w.) for two days or with their combinations. The hOGG1 modified comet assay revealed kidneys and liver oxidative DNA damage in OTA + CTN treated animals, which was not reversed by RSV. CTN did not reduce glutathione (GSH) or increase malondialdehyde (MDA) concentration in any tissue, while OTA reduced kidneys GSH and increased kidneys and liver MDA. RSV increased GSH concentrations in all tissues and decreased MDA concentration in the liver only. Oxidative stress is involved in the toxicity of OTA and CTN but it seems that it differs significantly in organs. Most of the effects on GSH and MDA in combined toxicity may be attributed to the toxic effects of OTA. RSV was effective in restoring the depleted GSH in all tissues but had no effect on the MDA concentration and DNA damage.

Sense and antisense peptides, i.e. peptides specified by complementary DNA and RNA sequences, interact with increased probability. Biro, Blalock, Mekler, Root-Bernstein and Siemion investigated the recognition rules of peptide—peptide interaction based on the complementary coding of DNA and RNA sequences in 3′ → 5′ and 5′ → 3′ directions. After more than three decades of theoretical and experimental investigations, the efficiency of this approach to predict peptide—peptide binding has been experimentally verified for more than 50 ligand—receptor systems, and represents a promising field of research. The natural genetic coding algorithm for sense and antisense peptide interactions combines following elements: of amino acid physico- chemical properties, stereochemical interaction, and bidirectional transcription. The interplay of these factors influences the specificity of sense—antisense peptide interactions, and affects the selection and evolution of peptide ligand—receptor systems. Complementary mRNA codon—tRNA anticodon complexes, and recently discovered Carter- Wolfenden tRNA acceptor-stem code, provide the basis for the rational modeling of peptide interactions based on their hydrophobic and lipophilic amino acid physico-chemical properties. It is shown that the interactions of complementary amino acid pairs according to the hydrophobic and lipophilic properties strongly depend on the central (second) purine base of the mRNA codon and its pyrimidine complement of the tRNA anticodon. This enables the development of new algorithms for the analysis of structure, function and evolution of protein and nucleotide sequences that take into account the residue's tendency to leave water and enter a nonpolar condensed phase considering its mass, size and accessible surface area. The practical applications of the sense—antisense peptide modeling are illustrated using different interaction assay types based on: microscale thermophoresis (MST), tryptophan fluorescence spectroscopy (TFS), nuclear magnetic resonance spectroscopy (NMR), and magnetic particles enzyme immunoassay (MPEIA). Various binding events and circumstances were considered, e.g., in situations with—short antisense peptide ligand (MST), L- and D-enantiomer acceptors (TFS), in low affinity conditions (NMR), and with more than one antisense peptide targeting hormone (MPEIA).

Perinatal hypoxia-ischemia is a specific and important pathological event in neonatal care practice. The data on relationship between the concentrations of cytokines in blood and cerebrospinal fluid (CSF) and perinatal brain injury are scarce. The aim of this study is to evaluate changes in interleukin (IL-1β, IL-6, and IL-18) and tumor necrosis factor alpha (TNF-α) levels in newborns with perinatal hypoxia (PNH). CSF and serum samples of 35 term and near-term (35-40 weeks) newborns with PNH, at the age of 3-96 hours, were analyzed using enzyme-linked immunosorbent assay. Control group consisted of 25 non-asphyxic/non-hypoxic infants of the same age sampled for clinically suspected perinatal meningitis, but proven negative and healthy otherwise. The cytokine values in CSF and serum samples were determined in relation to initial hypoxic-ischemic encephalopathy (HIE) staged according the Sarnat/Sarnat method, and compared with neurological outcome at 12 months of age estimated using Amiel-Tison procedure. The concentrations of IL-6 and TNF-α in serum of PNH patients were significantly higher compared to control group (p = 0.0407 and p = 0.023, respectively). No significant difference between average values of cytokines in relation to the stage of HIE was observed. Significantly higher levels of IL-6 and IL-18 corresponded to a mildly abnormal neurological outcome, while higher levels of IL-6 and TNF-α corresponded to a severely abnormal neurological outcome, at 12 months of age. Elevated serum levels of IL-6 and TNF-α better corresponded with hypoxia/ischemia compared to CSF values, within 96 hours of birth. Also, higher serum levels of IL-6, TNF-α, and IL-18 corresponded better with abnormal neurological outcome at 12 months of age, compared to CSF values.

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.

In vitro study of the effect of different volumes of twelve essential oils on the mycelial growth of economically significant phytopathogenic fungi (Fusarium oxysporum and Botrytis cinerea) and it was compared to the effect of a fungicide. The antifungal activity of essential oils is decreased with the duration of incubation and it differs depending on the type of phytopathogenic fungus and the applied volume. The most effective antifungal effect on both tested fungi was in the essential oil of thyme, with lowest values of IC50 while the weakest effect was in essential oils of eucalyptus and lemon, with the highest values of IC50. Certain essential oils, when applied in certain volumes, had the same or even better effect on the inhibition of the growth of mycelium when compared to the tested fungicides.

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.

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.

The primary objective of the study was to assess the vitamin D (VD) status of patients suffering from ITP. Children from the case cohort (total 21) were recruited from chronic ITP patients (followed as outpatients) and newly diagnosed ITP (prospective study) patients. VD deficiency (values o75 nmol/L) was detected in 11 patients with newly diagnosed ITP, and seven patients with chronic ITP. Only three patients with newly diagnosed, and none with chronic ITP had normal VD values. Newly diagnosed ITP patients had statistically significantly higher values (P o.044) of VD than the patients with chronic type of ITP. Platelets values did not follow VD level. VD deficiency is very common in children with either newly diagnosed or chronic ITP form. Therefore there is a utility supplementing VD in these patients. To investigate the role of VD as an immune modulating drug for patients with ITP, a prospective randomized placebo-controlled trial needs to be performed.

The difference between amino acid chemical properties that correlate to the exchangeability of protein sequence residues is often analysed using approach proposed by Grantham (1974). His difference formula, i.e., matrix, for calculating the distances between amino acid pairs of the protein consists of three essential amino acid physicochemical properties – composition, polarity and volume, that are significantly correlated to the substitution frequencies of the protein residues. Miyata et al. (1979) re-evaluated this concept, and showed that the degree of amino acid difference is just as adequately explained by only two physicochemical factors, volume and polarity. Miyazawa-Jernigan relative partition/hydrophobic energies (ε = Δ e ir), and Carter-Wolfenden vapor-to-cyclohexane scale ( G v>c = ΔG v>c ) are two alternative amino acid physicochemical parameters that are strongly correlated to their polarity and volume/mass, respectively. We show that the Miyazawa-Jernigan residue contact potential could be used instead of the Grantham polarity and composition parameters to derive an updated Miyata matrix. This substitution permits Miyata matrix correction for the amino acid parameters of: contact energies, repulsive packing energies, secondary structure energies, and Grantham’s composition property. Distance values calculated between both (classic and updated) Miyata matrices exhibit a strong correlation of r = 0.91. The possibility of analyzing residue distances based on Carter-Wolfenden water-to-cyclohexane (w > c) and vapor- to-cyclohexane (v > c) scales instead of the amino acid polarity and volume parameters is also discussed, and a new distance matrix is derived.

Background: Sputum induction is a method used over the years to investigate airway inflammation in patients with asthma and chronic obstructive pulmonary disease (COPD), to establish correct disease phenotype. Sputum production and expectoration are induced by inhaling hypertonic saline, which can also provoke adverse effects such as bronchoconstriction, lung hyperinflation, and dyspnea. The aim of this retrospective study was to assess the tolerability and safety of sputum induction in patients with more severe COPD stages. Methods: A total of 76 sputum inductions were performed in 20 patients with moderate or severe COPD (median FEV1 1.6 L 95%CI 1.47-1.71, or 51% 95% CI 47-54% of predicted) over a period of 32 days. Unique advantage of this study is multiple sputum induction in same patient. After premedication with 200 µg salbutamol, subjects inhaled increasing concentrations of aerosolized saline (0.9%, 3%, 4% and 5%), each for 7 minutes. FEV1 was recorded prior to induction and after each inhalation interval. Results: The median fall of FEV1 at the end of sputum induction was 127 mL (95%CI 90-176 mL) or 10% (95%CI 7-11%) from baseline post-bronchodilator value. In 11 cases (14%) the procedure was terminated due to an excessive bronchoconstriction (fall in FEV1>20% from baseline). None of the subjects reported adverse events. Conclusions: Sputum induction is safe and well tolerated by patients with moderate to severe COPD, which supports its use in clinical and research practice. Patients with a more severe airway obstruction may have a higher risk of excessive bronchoconstriction, precaution measures should be anticipated in those patients.

The classic results by Biro, Blalock and Root-Bernstein link genetic code nucleotide patterns to amino acid properties, protein structure and interaction. This study explores the use of the Miyazawa-Jernigan residue contact potential in analyses of protein interaction and recognition between sense and complementary (antisense) peptides. We show that Miyazawa-Jernigan residue contact energies, derived from 3D data, define the recognition rules of peptide-peptide interaction based on the complementary coding of DNA and RNA sequences. The model is strongly correlated with several other chemoinformatic scales often used for the determination of protein antigenic sites and transmembrane regions (Parker et al. r = 0.94 ; Rose et al. r = − 0.92 ; Manavalan-Ponnuswamy r = − 0.92 ; Cornette et al. r = − 0.91 ; Kolaskar-Tongaonkar r = − 0.91 ; Grantham r = 0.90 ; White-Wimley (octanol) r = − 0.88 ; Kyte-Doolittle r = − 0.85). The algorithms presented have important biomedical and proteomic applications related to modulation of the peptide-receptor function and epitope-paratope interaction, the design of lead compounds and the development of new immunochemical assays and diagnostic procedures.

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 β-melanocortin (β-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 β-MSH were used: 0.125, 0.250 and 0.500 mg/kg. Our results suggest that β-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 β-MSH at a dose of 0.250 mg/kg. The results justify further research on β-MSH peptide and its derivates in the inflammatory gastrointestinal diseases, and point out the possibility of using β-MSH in studies of digestive system pharmacology.

Proteins and peptides in mammals are based exclusively on L-amino acids. Recent investigations show that D-amino acids exhibit physiological effects in vivo, despite of their very small quantities. We have investigated the hepatoprotective effects of the Land D-enantiomers of alpha-melanocortin peptide (alpha-MSH). The results showed that peptide-enantiomerism is related to the protective effects of melanocortin peptides in vivo. L-alpha-MSH exhibited potent hepatoprotective effect in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice, while its D-mirror image was inefficient. Furthermore, the antibody to the L-peptide did not recognize the D-structure. The results indicate that the opposite peptide configuration may be used to modulate its function and metabolism in vivo and in vitro.

Background We investigated the possible non-lipid effects of simvastatin (SIMV) on paraoxonase 1 (PON1) and butyrylcholinesterase (BuChE) activity, as well as on malondialdehyde (MDA) levels in normolipidemic rats. Methods Two experimental groups of Wistar rats (10 mg/kg/day of SIMV) and two control groups (saline) underwent a 21-day treatment period (TP). On the 22nd day one experimental and one control group of rats were sacrificed. Remaining groups of animals were sacrificied on the 32nd day of the study (10-day after-treatment period (AT)). Blood samples and slices of liver, heart, kidney, and brain tissue were obtained for the measurement of PON1 and BuChE activity and levels of MDA. Data were analyzed by means of t -test for independent samples. p values ≤ 0.05 were considered as statistically significant. Results SIMV caused a significant decrease of serum and liver PON1 activity (18–24%, p ≤ 0.05) and MDA concentrations in the plasma, heart, liver, kidney, and brain (9–40%, p ≤ 0.05), while plasma and liver BuChE activity increased by 29% ( p ≤ 0.05) and 18%, respectively. All effects of SIMV were largely diminished following AT. The exception was MDA, which remained significantly decreased in plasma and all tissues analyzed. Conclusion SIMV significantly decreased PON1 activity and MDA levels and increased BuChE activity. We suggest that the decrease of MDA levels is a beneficial therapeutic effect of SIMV, for example in cardiovascular disorders, while the increase of BuChE activity, especially in brain, may be a potential adverse effect in patients with Alzheimer disease.

We present binary coding algorithm for the α- and β-protein fold prediction. The method links amino acid molecular polarity patterns and physicochemical properties of nucleotide bases coded by means of a binary addresses. Primary sequences that define secondary protein structure were analyzed with respect to the symbolic oligopeptides (SO) obtained by the reduction of the 20 amino acid letter alphabet into a binary alphabet of nonpolar group 0 (W, C, I, F, M, V, L, Y) and polar group 1 (Q, R, H, K, N, E, D, S, G, T, A, P). The groups were extracted from the Grantham polarity scale with the clustering around medoids procedure. The transformation of protein strings into binary coding patterns of the polar and nonpolar amino acid groups reduced analyzed elements within the protein motif of length n by the factor of 10n. SMO learning algorithm for the support vector machines was applied to classify α-helices and β-strands. It was shown that the relative frequencies of binary hexapeptides classify all 174 nonhomologous α- and β-protein folds from the Jpred database with 100% accuracy. The results of 10-fold cross-validation and leave-one-out test were 86.78%. Classification tree confirmed the results of SMO analysis and correctly classified 100% of the folds by means of 9 binary hexapeptides. Linear block triple-check code was proposed for the description of hexapeptide patterns. The presented method enables simple, quick, and accurate prediction of α- and β-protein folding types from the primary amino acid and nucleotide sequences on a personal computer. Our results imply that few amino acid polarity patterns specified by the nucleotide physicochemical properties describe basic protein folding types with >90% accuracy. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem, 2003

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 &gt, 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.

The effects of the gastric pentadecapeptide BPC 157, administered topically or systemically in burned mice were investigated. This agent is known to have a beneficial effect in variety models of gastrointestinal lesions, as well as on wound or fracture healing, also when given locally. Deep partial skin thickness burns (1.5cmx1.5cm) covering 20% of total body area, were induced under anesthesia on the back of the mice by controlled burning and gastric lesions were assessed 1, 2, 3, 7, 14 and 21 days following injury. The first application was immediately following burning, and thereafter, once daily, last 24h before sacrifice. In the initial experiments, exposition to direct flame for 5 seconds, the BPC 157 was applied at 10 mg or 10 ng/kg b.w. i.p. by injection or alternatively, topically, at the burn, as a thin layer of cream (50 mg of BPC 157 dissolved in 2 ml of distilled water was mixed with 50 g of commercial neutral cream (used also as local vehicle-control)), while silver sulfadiazine 1% cream was a standard agent acting locally. Others received no local medication: they were treated i.p. by injection of distilled water (distilled water-control) or left without any medication (control). In subsequent experiments involving deeper burns (direct flame for 7 seconds), BPC 157 creams (50 ľg, 5 ľg, 500 ng, 50 ng or 5 ng of BPC 157 dissolved in 2 ml of distilled water was mixed with 50 g of commercial neutral cream), or vehicle as a thin layer of cream, were applied topically, at the burn. Compared with untreated controls, in both experiments, in the BPC 157 cream treated mice all parameters of burn healing were improved throughout the experiment: less edema was observed and inflammatory cells number was decreased, less necrosis, an increased number of capillaries along with an advanced formation of dermal reticulin and collagen fibers and an increased number of preserved follicles were observed. Two weeks after injury, BPC 157 cream-treated mice completely reversed the otherwise poor reepithelization ratio noted in the untreated control or mice treated with only cream vehicle. Tensiometry investigation showed an increased breaking strength and relative elongation of burned skin, while water content in burned skin decreased. This was, however, not the case with the vehicle cream or silver sulfadiazine cream-mice. Relative to the control values, in silver sulfadiazine cream-treated mice, only collagen fiber formation was increased, in addition to a decreased inflammatory cells number. Relative to control values, BPC 157 given intraperitoneally decreased the number of inflammatory cells, lowered water content in burned skin, and raised breaking strength and relative elongation of burned skin during tensiometry. Through the experimental period gastric lesions were continously noted in all thermally injured mice left without local medication and they were consistently attenuated only by BPC 157 treatments: either given intraperitoneally (at either dose), or given locally (at either concentrations). Other treatments (i.e., local treatment with silver sulfadiazine cream or neutral cream in mice subjected for 5 seconds to direct flame), lead to only poor, if any attenuation. This stable gastric pentadecapeptide appears to be active and give a stimulation to burn healing at the defect site. The agent may act by causing an upregulation of the growth factors, as well as influences other local factors.

Gastrectomy often results in increased likelihood of osteoporosis, metabolic aberration, and risk of fracture, and there is a need for a gastric peptide with osteogenic activity. A novel stomach pentadecapeptide, BPC-157, improves wound and fracture healing in rats in addition to having an angiogenic effect. Therefore, in the present study, using a segmental osteoperiosteal bone defect (0.8 cm, in the middle of the left radius) that remained incompletely healed in all control rabbits for 6 weeks (assessed in 2 week intervals), pentadecapeptide BPC-157 was further studied (either percutaneously given locally [10 microg/kg body weight] into the bone defect, or applied intramuscularly [intermittently, at postoperative days 7, 9, 14, and 16 at 10 microg/kg body weight] or continuously [once per day, postoperative days 7-21 at 10 microg or 10 ng/kg body weight]). For comparison, rabbits percutaneously received locally autologous bone marrow (2 mL, postoperative day 7). As standard treatment, immediately after its formation, the bone defect was filled with an autologous cortical graft. Saline-treated (2 mL intramuscularly [i.m.] and 2 mL locally into the bone defect), injured animals were used as controls. Pentadecapeptide BPC-157 significantly improved the healing of segmental bone defects. For instance, upon radiographic assessment, the callus surface, microphotodensitometry, quantitative histomorphometry (10 microg/kg body weight i.m. for 14 days), or quantitative histomorphometry (10 ng/kg body weight i.m. for 14 days) the effect of pentadecapeptide BPC-157 was shown to correspond to improvement after local application of bone marrow or autologous cortical graft. Moreover, a comparison of the number of animals with unhealed defects (all controls) or healed defects (complete bony continuity across the defect site) showed that besides pentadecapeptide intramuscular application for 14 days (i.e., local application of bone marrow or autologous cortical graft), also following other pentadecapeptide BPC-157 regimens (local application, or intermittent intramuscular administration), the number of animals with healed defect was increased. Hopefully, in the light of the suggested stomach significance for bone homeostasis, the possible relevance of this pentadecapeptide BPC-157 effect (local or intramuscular effectiveness, lack of unwanted effects) could be a basis for methods of choice in the future management of healing impairment in humans, and requires further investigation.

Background: A novel gastric pentadecapeptide, BPC 157, has been shown to attenuate different lesions (i.e., gastrointestinal tract, liver, pancreas, somatosensory neurons). This suggests an interaction with the dopamine system. When used alone, BPC 157 does not affect gross behavior or induce stereotypy. Methods: We first investigated the effect of pentadecapeptide BPC 157 on stereotypy and acoustic startle response in rats, given as either a prophylactic (10 μg/kg IP) or therapeutic (10 ng/kg IP) regimen, with the dopamine indirect agonist amphetamine (10 mg/kg IP). Results: There was a marked attenuation of stereotypic behavior and acoustic startle response. When the medication was given at the time of maximum amphetamine-induced excitability, there was a reversal of this behavior. A further focus was on the effect of this pentadecapeptide on increased climbing behavior in mice pretreated with the dopamine antagonist haloperidol (5.0 mg/kg IP), and subsequently treated with amphetamine (20 mg/kg IP challenge 1, 2, 4, and 10 days after haloperidol pretreatment). This protocol is usually used for the study of behavioral supersensitivity to the amphetamine stimulating effect. Conclusions: An almost complete reversal was noted when pentadecapeptide was coadministered with haloperidol. Together, these data provide compelling evidence for the interaction of pentadecapeptide BPC 157 with the dopamine system.

The 15 amino acid agent BPC 157, showing a wide range of organoprotective action in different experimental models, was used in our experiments in order to establish its influence on different elements connected with the healing process. Elements thought to be of greatest importance in the process of healing are formation of granulation tissue, angiogenesis and production of collagen. In our work we tested the influence of BPC 157 on: granulation tissue and collagen formation, on angiogenesis as well as on tensile strength development, using three experimental rat models: 1) skin incisional wounds ; 2) colon-colon anastomoses ; and 3) angiogenesis model with synthetic sponge implantation. The specimens were histologically assessed for collagen, reticulin and blood vessels using scoring and morphometry. In all experiments significant differences between BPC 157-treated animals and controls were found, showing a strong, promoting involvement of BPC in the healing process. It is worth noting that these effects were achieved by different routes of application, including intragastric and local, making BPC 157 a potentially useful therapeutic agent.

A superior effectiveness in various lesionassays was noted for the novel pentadecapeptide BPC 157,originated from human gastric juice protein (BPC) andclaimed to be a cytoprotective agent. From this viewpoint, as a previously untreatedexperimental improvement to create an acid-freeenvironmental for cytoprotection studies, totalgastrectomy was done 24 hr before the ulcerogenicprocedure. In the absence of stomach and gastric acid, the damagingeffects of cysteamine (400 mg/kg subcutaneously, death24 hr thereafter), to date thought to be an acid-relatedduodenal ulcerogen, and the BPC 157 cytoprotective effect (10 mug or 10 ng/kg intraperitoneally)were further challenged. BPC 157 was compared withreference agents [cimetidine (50), ranitidine (10),omeprazole (10), bromocriptine (10) and atropine (10) (mg/kg intraperitoneally, 1 hr beforecysteamine] known to be also cytoprotective. In naiverats, with intact stomach, all of them showed a strongbeneficial effect. Interestingly, in gastrectomizedanimals, the application of BPC 157 or the referenceagents before cysteamine significantly prevented theotherwise severe duodenal lesion development noted inthe control gastrectomized cysteamine rats. In groups without cysteamine, no lesions were noted(laparotomy, gastrectomy only, 24 or 48 hr postsurgicalperiod), nor was lesion potentiation seen incysteamine-treated laparotomized animals. In summary,these findings -- equal damaging effect ofcysteamine and equal protection of pentadecapeptide BPC157 and reference agents in gastrectomized and rats withintact stomach -- seem to be particularly relevantfor a cytoprotective viewpoint. Without a stomach,the cysteamine damaging effect was convincingly definedas an essential gastric acid-independent injury(analogous to ethanol gastric lesions). Likewise, a high “cytoprotective capacity,”apparently acid independent, common for all testedagents (novel pentadecapeptide BPC 157, cimetidine,ranitidine, omeprazole and atropine) could be clearlystressed.

BORIS is a paralog of a highly conserved, multi-functional chromatin factor CTCF. Unlike CTCF, which has been shown to possess tumor-suppressive properties, BORIS belongs to the "cancer/testis antigen" family normally expressed only in germ cells and aberrantly activated in a variety of tumors. The consequences of BORIS expression, relative abundance of its isoforms, and its role in carcinogenesis have not been completely elucidated. It activates transcription of hTERT and MYC, genes relevant for laryngeal carcinoma progression. In this study, BORIS expression has been analyzed at the transcriptional level by RT-PCR and protein level by semi-quantitative immunohistochemistry in 32 laryngeal squamous cell carcinomas and adjacent non-tumorous tissue. BORIS was detected in 44 % (14/32) laryngeal squamous cell carcinoma samples, while it was detected only in one normal, tumor-adjacent tissue sample. Tree based survival analysis, using the recursive partitioning algorithm mvpart, extracted the ratio of relative abundance of BORIS transcript variants containing exon 7 (BORIS 7+) and those lacking exon 7 (BORIS 7-) as an independent prognostic factor associated with disease relapse during a 5-year follow-up period. Patients having BORIS 7+/BORIS 7- ratio ≥1 had a higher rate of disease relapse than patients with BORIS 7+/BORIS 7- ratio