Frequency of main western-Euroasian mtDNA haplogroups and paleolithic and neolithic lineages in the genetic structure of population of northeastern Bosnia.

Mitochondrial DNA (mtDNA) variations were analyzed in a sample of 245 individuals of Bosnian-Herzegovinian population from the area of Northeastern Bosnia (also known as Tuzla region). Haplogroup affiliation was determined using RFLP method (Restriction Fragment Length Polymorphism) analyzing haplogroup-specific markers of mtDNA coding region, characteristic for the main Western-Eurasian haplogroups. Additional analyses of two sequenced hypervariable segments (HVSI and HVSII) of mtDNA control region were performed in order to identify U subhaplogroups. The study revealed that 95.51% of the analyzed individuals belonged to the typical Western-Eurasian haplogroups: H, I, J, K, T U, V, W or X. The most frequent haplogroup in the analyzed population was the haplogroup H (52.65%) which, due to its increased frequency, represents a marking haplogroup of the population of Northeastern Bosnia. The results of intergroup genetic analysis showed that Bosnian-Herzegovinian population is genetically closer to previously studied populations of Herzegovinians (part of Bosnia and Herzegovina), Slovenians and Croats in relation to other neighboring populations located in Southeastern Europe. Our study also suggests that population genetic structure of Tuzla region is dominated by mutations that are classified as "Paleolithic". These mutations were probably brought to the area of northeastern Bosnia through waves of prehistoric and historic migrations, but the impact of any pre-Neolithic, Neolithic or some "later" migrations, with a slightly lower contribution to the genetic structure of this population, also cannot be neglected.

Geranium robertianum L. tolerates various soil types burdened with heavy metals.

Many heavy metals (HMs) are essential micronutrients for the growth and development of plants. However, human activities such as mining, smelting, waste disposal, and industrial processes have led to toxic levels of HMs in soil. Fortunately, many plant species have developed incredible adaptive mechanisms to survive and thrive in such harsh environments. As a widespread and ruderal species, Geranium robertianum L. inhabits versatile soil types, both polluted and unpolluted. Considering the ubiquity of G. robertianum, the study aimed to determine whether geographically distant populations can tolerate HMs. We collected soil and plant samples from serpentine, an anthropogenic heavy metal contaminated, and a non-metalliferous site to study the physiological state of G. robertianum. HMs in soil and plants were determined using flame atomic absorption spectrometry. Spectrophotometric methods were used to measure the total content of chlorophylls a and b, total phenolics, phenolic acids, flavonoids, and proline. Principal component analysis (PCA) was used to investigate the potential correlation between HMs concentrations gathered from various soil types and plant samples and biochemical data acquired for plant material. A statistically significant difference was observed for all localities regarding secondary metabolite parameters. A positive correlation between Ni and Zn in soil and Ni and Zn in plant matter was observed (p<0.0005) indicating higher absorption. Regardless of high concentrations of heavy metals in investigated soils, G. robertianum displayed resilience and was capable of thriving. These results may be ascribed to several protective mechanisms that allow G. robertianum to express normal growth and development and act as a pioneer species.

Antioxidative and antitumor properties of in vitro-cultivated broccoli (Brassica oleracea var. italica).

CONTEXT: Broccoli [Brassica oleracea L. var. italica Plenck. (Brassicaceae)] contains substantial quantities of bioactive compounds, which are good free radical scavengers and thus might have strong antitumor properties. Enhancing production of plant secondary metabolites could be obtained with phytohormones that have significant effects on the metabolism of secondary metabolites. In that manner, in vitro culture presents good model for manipulation with plant tissues in order to affect secondary metabolite production and thus enhance bioactive properties of plants. OBJECTIVE: Estimation of the antioxidative and antitumor properties of broccoli cultivated in different in vitro conditions. MATERIALS AND METHODS: In vitro germinated and cultivated broccoli seedlings, as well as spontaneously developed calli, were subjected to Soxhlet extraction. Antioxidative activity of the herbal extracts was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH(•)) radical method. Antitumor properties of the extracts were determined using crown-gall tumor inhibition (potato disc) assay. RESULTS: Three, 10, 20, and 30 days old broccoli seedlings, cultivated in vitro on three different Murashige-Skoog media, two types of callus, and seedlings from sterile filter paper were used for extraction. In total, 15 aqueous extracts were tested for antioxidative and antitumor potential. Three day-old seedlings showed the highest antioxidative activity. Eleven out of 15 aqueous extracts demonstrated above 50% of crown-gall tumor inhibition in comparison with the control. Tumor inhibition was in association with types and concentrations of phytohormones presented in growing media. DISCUSSION AND CONCLUSIONS: It is demonstrated that phytohormones in plant-growing media could affect the bioactive properties of broccoli either through increasing or decreasing their antioxidative and antitumor potential.

Effects of dipotassium trioxohydroxytetrafluorotriborate (K2[BO3F4OH]) on genetic material and inhibition of cell division in human cell cultures.

We have examined antiproliferative, cytotoxic, and genotoxic potential of a halogenated boroxine dipotassium trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]). The impact on cell growth was evaluated by alamarBlue assay in basal cell carcinoma culture. Cytostatic, cytotoxic, and genotoxic potential were evaluated in lymphocytes culture, applying cytokinesis-block micronucleus cytome assay and chromosome aberrations analysis. Tested concentrations (0.05, 0.1, 0.2, and 0.4 mg/mL) were correlated with inhibition of cell growth in basal cell carcinoma culture and with the lymphocytes proliferation. Clastogenic activity has been confirmed, without evidences of aneugenic activity, in human lymphocytes.

Tannerella forsythia detected in infected root canals using nested PCR.

PURPOSE: This study assessed the occurrence of Tannerella forsythia in patients with acute and chronic primary endodontic infections. METHODS: Clinical samples were collected from 40 patients with acute and chronic periradicular disease. Nested polymerase chain reaction (PCR) assay technique was used to detect the presence of T. forsythia in primary endodontic infections. The first round of PCR amplification used universal primers to detect the 16S rDNA sequence. Product from the first round was then used to amplify T. forsythia specific fragment with species-specific pairs of primers. RESULTS: T. forsythia was found in 12 of 27 chronic and 5 of 13 acute infected patients for an overall occurrence frequency of 42.5%. No significant correlation was found between patients with the T. forsythia positive genotype and the occurrence of clinical symptoms in the primary endodontic infections (P < 0.05) (P = 0.496). Also, no significant relationship was found between the occurrence of T. forsythia and the patient's age (P = 0.61) or gender (P = 0.239).

Identification of skeletal remains of Communist Armed Forces victims during and after World War II: combined Y-chromosome (STR) and MiniSTR approach.

AIM: To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. METHODS: Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Skofja Loka area (Lovrenska Grapa and Zolsce) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpF/STR MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. RESULTS: Following the Y-STR analysis, 1 of the "weak matches" previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Zolosce, we were able to obtain 6 useful DNA profiles. CONCLUSION: The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and mini-STR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II.

Overview of human population-genetic studies in Bosnia and Herzegovina during the last three centuries: history and prospective.

Modern Bosnia and Herzegovina is a multi-ethnic and multi-religion country, with a very stormy history. Certain archaeological findings indicate continuous population of its territory since the Paleolithic. In time, vast number of different factors jointly influenced fascinating diversity of local human populations. A great number of small, more or less isolated, indigenous populations, make this area quite attractive for population-genetic surveys of different levels and approaches. Austro-Hungarian military physicians conducted the very first known bio-anthropological analyses of Bosnia-Herzegovina population at the end of the 19th century. Thus, the first step towards resolving the genetic structures of local B&H human populations was made. The studies that followed (conducted throughout most of the 20th century) were primarily based on the observation of various phenotypic traits. This stage was followed by the examination of various cytogenetic and fundamental DNA based molecular markers. The efforts undertaken over the last three centuries revealed "human genetic treasure" in Bosnia and Herzegovina. However, even now, after all the studies that were conducted, many interesting features remain to be discovered and described within the existing local human populations.

DNA identification of skeletal remains from the World War II mass graves uncovered in Slovenia.

AIM: To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Skofja Loka, Slovenia. METHODS: The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. Quantifiler Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. RESULTS: Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. CONCLUSION: Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project.

Allele frequencies for 15 short tandem repeat loci in a representative sample of Bosnians and Herzegovinians.

Allele frequencies for the 15 STR loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA) included in the PowerPlex 16 kit were obtained from a multiethnic sample of 100 unrelated individuals born in Bosnia and Herzegovina.

Population data at two short tandem repeat loci D2S1338 and D19S433 in the sample of multinational Bosnia and Herzegovina residents.

POPULATION: We have analyzed the distribution of allele frequencies at two short tandem repeats loci (D2S1338 and D19S433) in a multinational sample of Bosnia and Herzegovina (B&H) residents. A total of 110 unrelated male and female individuals (Caucasians) from different regions of B&H were sampled for the analysis. We ensured that the sample reflected approximate proportional participation of the three main ethnic groups in the population of B&H (Bosniacs-Muslim [45%], Serbs [34%], Croats [21%]).

Assessment of relatedness between neurocan gene as bipolar disorder susceptibility locus and schizophrenia.

Large scale genetic association meta-analyses showed that neurocan (NCAN) gene polymorphism rs1064395 is susceptibility locus for bipolar disorder. These studies also included patients with bipolar disorder originated from Bosnia and Herzegovina. Followed by theory of shared genetic elements between bipolar disorder and schizophrenia susceptibility, other studies explored several genetic factors with schizophrenia vulnerability as well. In this work, authors investigated the association between previously confirmed bipolar disorder genetic risk factor- neurocan with schizophrenia in a population sample of Bosnia and Herzegovina. Ethical aspects of this research were assessed by Ethics Committee of Clinical Center University of Sarajevo. Blood samples for DNA extraction were taken from the total of 86 patients and healthy individuals who previously signed informed consent. Genotyping for rs 1064395 was done using direct sequencing method. A case-control analysis of common genetic polymorphism within neurocan gene and schizophrenia status in a consecutively sampled patient cohort have been done using Fisher-exact test with odds-ratio calculation. No statistically significant allele and genotype association with disease status was found (p>0.05). Our finding supports the fact that large-scale genetic association studies approach need to be employed when detecting the variants with small additive effect in phenotypes with complex ethiology.

Antioxidant and antiproliferative activities of Helleborus odorus Waldst. & Kit, H. multifidus Vis. and H. hercegovinus Martinis.

This study was undertaken in order to evaluate possible antioxidative and antiproliferative activities of three Helleborus taxa. The dry leaves and roots of three Helleborus taxa were extracted with ethanol and water. A phytochemical evaluation of the selected extracts was performed using spectrophotometric methods and a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay was used for measuring the antioxidative activity of extracts. The antiproliferative activity of the three Helleborus taxa was studied using Burkitt's lymphoma B cells (BJAB) cell lines. The phytochemical evaluation showed that the leaves contain high levels of total phenolic and flavonoid content. Results from the DPPH assay indicated that the activity of the ethanol and water extracts of the leaves was higher than that of positive control (thymol). Extracts from the roots of H. odorus also displayed higher antioxidant activity than the positive probe, while H. mulifidus and H. hercegovinus root extracts were less effective. A statistically significant correlation between total phenolic content and antioxidative properties indicates that these compounds contribute to the antioxidant activity. The highest percentage of cell growth inhibition was observed when testing the water root extracts of H. multifidus (50.14%) and H. hercegovinus (49.04%). In contrast, the water leaf extract of H. hercegovinus exhibited the lowest inhibition of cell growth (8.59%), although it showed strong antioxidant activity.