Snakebites are a significant public health problem in many tropical and subtropical regions, causing extensive morbidity and mortality. Traditional snake antivenoms face multiple challenges, including allergenicity, high production costs, and logistical difficulties, highlighting the urgent need for new therapeutic approaches. This pilot study explores the potential of oligopeptides as therapeutic inhibitors targeting the neurotoxic sites of ammodytoxin A (AtxA; PDB: 3G8G) from Vipera ammodytes. We selected two sense oligopeptides to represent critical neurotoxic regions of AtxA as targets for inhibition by complementary antisense peptides. Utilizing a heuristic antisense peptide design based on the molecular recognition theory, we modeled two antisense oligopeptides as complementary counterparts for each sense oligopeptide. The modeled sense and antisense peptides were commercially synthesized, and their binding affinities were evaluated using spectrofluorometric titrations. The determined dissociation constants (KD) were in the range of 1–10 μM for all sense–antisense pairs, revealing relatively strong binding affinities. Confirmation of sense–antisense peptide binding prompted further investigation into their potential binding to the native target protein through global docking simulations using the HPEPDOCK web server. The results highlight the applicability of molecular recognition theory in the development of antisense peptides that could change therapeutic strategies in various biomedical fields. Further studies are needed to investigate the therapeutic efficacy and broader applications of these peptides.

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (phi(ST) = 0.190) and fully CpG methylated (phi(ST) = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (phi(ST) = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

Background:Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation.Methods:Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively.Results:We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19+ B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly.Conclusions:Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3+ T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.

A convenient spectroelectrochemical method for the determination of holo-transferrin reduction potential is described. All materials and accessories are commercially available and the results are in agreement with previous reports that required custom-built hardware. The method requires minimal preparation with simple sample handling, small solution volume, cost-effective electrode replacement and automated measurement. This method can easily be applied to the study of similar systems in aqueous media and might be of particular importance in the momentous field of glycomics, where large numbers of protein isoforms need to be characterized.

Short-toothed sage (Salvia brachyodon Vandas) is Illyrian-Adriatic stenoendemic and one of the rarest plant species of the Dinaric karst. S. brachyodon grows in Croatia and Bosnia and Herzegovina/Montenegro and only three localities have been confirmed at the present time: (1) Mt. Zmijino brdo (Sv. Ilija) near Orebic on the peninsula of Peljesac, Croatia, (2) Mt. Konavoska brda near Velji do, north of Cavtat in Konavle region, Croatia, and (3) Mt. Orjen, near Vrbanje, at the border of Bosnia and Herzegovina and Montenegro. It has been categorized as near thretened (NT) species in Croatia and as endangered (EN) in Montenegro. The habitats of S. brachyodon greatly differ among localities and the distinctions at the morphological and molecular levels may represent the adaptation of the plant to the specific habitat conditions. Heritable epigenetic variation could influence the course of evolution in plants, as it can affect the processes of adaptation and divergence through selection of stable epigenetic variants without involvement of genetic variation. In order to understand the true importance of epigenetic processes in a stenoendemic plant of a very narrow range size existing epigenetic variation has been assessed and compared to genetic variation. The aim of this research was to determine genetic and epigenetic diversity and differentiation of the three known short- toothed sage populations by using molecular markers. Microsatellite markers (SSRs) and amplified fragment-length polymorphysims (AFLPs) were used to investigate the genetic diversity and structure within and among sampled natural plant populations while the methylation-sensitive amplified polymorphisms (MSAPs) were used to assess the epigenetic diversity.

Cycling cells of Quercus robur have a simple nuclear organization where most of the heterochromatin is visible as DAPI-positive chromocenters, which correspond to DAPI bands at the (peri)centromeric region of each of the 24 chromosomes of the oak complement. Immunofluorescence using 5-mC revealed dispersed distribution of the signal throughout the interphase nucleus/chromosomes without enrichment within DAPI-positive chromocenters/bands, suggesting that DNA methylation was not restricted to constitutive heterochromatin, but was associated with both euchromatic and heterochromatic domains. While H3K9ac exhibited typical euchromatin-specific distribution, the distributional pattern of histone methylation marks H3K9me1, H3K27me2, and H3K4me3 showed some specificity. The H3K9me1 and H3K27me2, both heterochromatin-associated marks, were not restricted to chromocenters, but showed additional dispersed distribution within euchromatin, while H3K27me2 mark also clustered in foci that did not co-localize with chromocenters. Surprisingly, even though H3K4me3 was distributed in the entire chromatin, many chromocenters were enriched with this euchromatin-specific modification. We discuss the distribution of the epigenetic marks in the context of the genome composition and lifestyle of Q. robur.

18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic spacer (IGS) that contains transcription initiation/termination signals and usually repeating elements. In this study, we performed for the first time thorough sequence analysis of rDNA IGS region in two dominant European oaks, Quercus petraea and Q. robur, in order to investigate (1) if IGS sequence composition allows discrimination between these two species, and (2) if there is an rDNA length heterogeneity arising from IGS sequence. Two spacer length variants (slvs), 2 and 4 kb in length, were found in the genomes of both species. Inter-comparison of both slvs revealed no species-specificity in sequence or structural organization. Both slvs could be divided into four subregions; (1) the subrepeat region containing three repeated elements, (2) the AT-rich region containing matrix attachment sites and putative origin of replication, (3) the promoter region containing putative transcription initiation site and (4) the 5'ETS region. In the 4-kb slvs all four subregions are extended, and the subrepeat, AT-rich and promoter regions are duplicated. This is unique compared to other known IGS sequences where the variation in number of subrepeats is responsible for slvs creation. We also propose a possible evolutionary scenario to explain the formation of the subrepeat region in oak IGS. Results obtained in this work add to the previous picture of low-genetic differentiation of the two oaks and provide important data for further analyses of the function of IGS in control of rRNA gene expression.

Aiming to evaluate the effects of population substructure on the reliability of a DNA correspondence in the process of human identification, we used the model of "in silico" constructed populations with and without substructure. Effects of population substructure were evaluated at the level of locus heterozygosity, Hardy-Weinberg equilibrium and mini-haplotype distribution. Inbreeding in a subpopulation of 100 individuals through 10 generations did not significantly alter the level of heterozygosity and Hardy-Weinberg equilibrium. However, analysis of mini-haplotype distribution revealed a significant homogenization in separated subpopulations. Average observed mini-haplotype frequency (f(o)) increased to threefold from expected values (f(e)), and the number of mini-haplotypes with f(o)/f(e) above 10 increased over sixfold, suggesting that the effects of population substructure on calculated likelihood ratios (LR) might be larger than previously estimated. In most criminal cases, this would not represent a problem, whereas for identifications in large-scale mass fatality events, population substructure might considerably increase the risk of false identification.

Serpentine soils are characterized by extreme ecological conditions for plant development - they are barren of plant nutrients, contain high concentrations of heavy metals, they are shallow and do not hold water well, which results in dry and exposed habitats. The stresses induced in such conditions exert evolutionary pressure for selection of traits and mutations that allow certain plants to adapt to serpentine soils. Serpentine flora in general provides an opportunity to examine adaptive evolutionary mechanisms. Lilium bosniacum is an endemic species of Balkan Peninsula, which usually grows on limestone or dolomite substrates with deep soils rich in nutrients and at altitudinal optimum from 1200 m to 1300 m. We found a population of L. bosniacum growing on inclined slope of serpentine substrate at altitude of 970 m, at the environmental conditions that are not habitual for this species. This is the first serpentine population found in the distributional range of Bosnian lily. Cytogenetic and molecular analyses revealed variations in karyotype/genome features between serpentine and non-serpentine populations, as well as within serpentine population, in terms of changed chromosome morphology, different number and position of 18S-26S rDNA loci and CMA bands, different copy number and transcriptional activity of rRNA genes, presence/absence of B-chromosomes and different rDNA IGS length variants. Full sequence is given for IGS length variants. In addition, analysis of leaf anatomy revealed difference in number and size of stoma per leaf area between non-serpentine and serpentine populations. Changes of karyotype/genome and anatomic features found in serpentine population are discussed in respect to the extreme life conditions that are characteristic for serpentines.

Quinolones are an important group of powerful synthetic broad spectrum antimicrobial drugs which are active against gram-negative and gram-positive microorganisms as well as some atypical pathogens and anaerobes. This review contains information about historical development, chemical structures, mechanism of action and activity spectrum of these drugs. Therapeutic use, pharmacokinetics, common adverse effects, interactions and methods of determination in bodily fluids are also covered. Understanding of properties of individual agents in this group is necessary for appropriate and safe administration to each patient. Growing incidence of microbial resistance to these drugs calls for rational use in future.

Gangliosides are plasma membrane components with ceramide anchor and different number of sialic acid residues linked to inner or/and outer galactose of sugar core chain. Major forms appearing predominantly in nervous tissue are: GM1, GD1a, GD1b and GT1b. They are ten times more abundant in CNS of higher animals then in any extra-neuronal tissue. The exact physiological and neurobiological role of each form is still unknown, but there is significant evidence that they participate in neurogenesis, brain development and maturation, synapse formation, memory, communication and adhesion of cells and signal transduction. Gangliosides bind to physiological receptors (GD1a and GT1b are ligands for MAG in process of myelination) as well as external biotoxins. Aiming to understand the evolution of functional roles of gangliosides, we have studied the distribution of the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in brains of trout, carp, green frog, long-nosed viper, chicken, bat, rat, ferret, rabbit and cat. Highly specific monoclonal antibodies were used to detect ganglioside patterns in paraformaldehyde-fixed brains.

Background and purpose: The gender should be considered as a basic variable at all levels of biomedical and health related research. Thus, we tested the hypothesis whether gender and time of day affect tail pressure thresholds in Wistar rats. Materials and methods: 11 female and 11 male Wistar rats, in the age of 6 months were used. After two days of habituation period, tail pressure thresholds were determined twice a day (9.00-10.00 h and 15.00-16.00 h) in 3 consecutive days. Expressed in grams (g), the pain thresholds were measured by applying increasing force on the tail, until the tail was withdrawn as the result of pain. Data were analyzed using analysis of variance (ANOVA) and expressed as the mean p ; S.E.M. Results: The pain thresholds were significantly higher in male, as compared to female rats, when testing took place in the morning (days 1-3 ; F (5, 52) = 4.93 ; P<0.01). When testing took place in the afternoon, there was no difference in the pain threshold values between female and male rats (days 1-3 ; F (5, 52) = 2.19, not significant at P<0.05). Significant decrease in afternoon pain threshold values, as compared to morning values, was detected within male (days 1-3 ; F (5, 56) = 3.5 ; P<0.05), but not within female rats (days 1-3 ; F (5, 48) = 1.1 not significant at P<0.05). Conclusion: The results presented here indicate that both, gender and time of day within light phase of day cycle, affect tail pressure thresholds in Wistar rats.

AIM:To analyze statistically and logically the significance of genetic matches between skeletal remains and relatives of missing persons in the process of identification of war victims by DNA typing. METHODS:DNA was isolated from bone and blood samples and short tandem repeat (STR) loci were typed by using AmpFLSTR Profiler, Profiler Plus, and Identifiler kits. Novel mini-haplotype analysis that compares matches in all three-locus combinations of alleles was developed and used in the analysis of inbreeding in the group of 295 unrelated individuals. RESULTS:While comparing 98 skeletal remains exhumed in the process of identification of war victims in Croatia with over 3,000 genotypes of relatives of missing persons, we revealed 20 cases of 14-locus matches and 4 cases of 15-locus matches between unrelated people. We hypothesized that this unexpectedly high number of false matches might be a consequence of local inbreeding and supported this hypothesis with very low correlation between the probability of a genotype and the number of matching genotypes in the database (R(2) = 0.36). Further support for the hypothesis was obtained by the analysis of mini-haplotypes, which revealed up to 90% overrepresentation of some mini-haplotypes. CONCLUSIONS:STR DNA typing is the "golden standard" of human identification, but evidential value of a genetic match can be easily misinterpreted. Therefore, careful use of statistical methods is essential for the proper evaluation of laboratory results. Whenever possible, multiple relatives should be analyzed and other evidence based on the information about time, place, and other conditions of disappearance, as well as anthropological and other "classical" forensic data should always be put together and compared before any final decision about the identity is made.