Dinaric limestone cave systems, recognized as a hotspot of subterranean biodiversity, inhabit composite microbial communities whose structure, function and importance to ecosystems was poorly considered until the last few years. Filamentous microbial biofilms from three caves in Dinaric karst were assessed using 16S rRNA‐based phylogenetic approach combined with universally protein coding genes/proteins. Studied clone libraries shared divisions but phylogenetic distribution of the obtained phylotypes differed: in Veternica and Vjetrenica clone libraries, Nitrospirae prevailed with 36% and 60% respectively, while in Izvor Bistrac the most abundant were Alphaproteobacteria (41%) followed by Firmicutes (32%). Moreover, three phylotypes were associated with novel uncultured candidate divisions OP3, WS5 and OD1 revealing the diversity and uniqueness of the microbial world in caves. Deeply understanding subterranean habitats could elucidate many new aspects in phylogeny and evolution of microorganisms as well as animal taxa, adjacent to their energy suppliers in microbial communities and biofilms.

Modern Bosnia and Herzegovina is a multinational and multi-religious country, situated in the western part of the Balkan Peninsula in South-eastern Europe. According to recent archaeological fi ndings, Bosnia and Herzegovina has been occupied by modern humans since the Palaeolithic period. The structure of Bosnia-Herzegovina’s human populations is very complex and specifi c, due to which it is interesting for various population-genetic surveys. The population of Bosnia and Herzegovina has been the focus of bio-anthropological and population genetics studies since the 19th century. The fi rst known bio-anthropological analyses of Bosnia-Herzegovina population were primarily based on the observation of some phenotypic traits. Later examinations included cytogenetic and DNA based molecular markers. The results of all studies which have been done up to date showed no accented genetic difference among the populations (based on geographical regions) with quite high diversity within them. Human population of Bosnia and Herzegovina is closely related to other populations in the Balkans. However, there are still many interesting features hidden within the existing diversity of local human populations that are still waiting to be discovered and described.

BACKGROUND Periapical inflammatory lesions have been investigated previously, but understanding of pathogenesis of these lesions (granulomas and radicular cysts) at the molecular level is still questionable. Matrix metalloproteinases (MMPs) are enzymes involved in the development of periapical pathology, specifically inflammation and tissue destruction. To elucidate pathogenesis of periapical granulomas and radicular cysts, we undertook a detailed analysis of gene expression of MMP-1, MMP-2 and their tissue inhibitors, TIMP-1 and TIMP-2. METHODS A total of 149 samples were analyzed using real-time PCR (59 radicular cysts, 50 periapical granulomas and 40 healthy gingiva samples as controls) for expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 genes. The determination of best reference gene for expression analysis of periapical lesions was done using a panel of 12 genes. RESULTS We have shown that β-actin and GAPDH are not the most stable reference controls for gene expression analysis of inflammatory periapical tissues and healthy gingiva. The most suitable reference gene was determined to be SDHA (a succinate dehydrogenase complex, subunit A, flavoprotein [Fp]). We found that granulomas (n = 50) and radicular cysts (n = 59) exhibited significantly higher expression of all four examined genes, MMP-1, MMP-2, TIMP-1, and TIMP-2, when compared to healthy gingiva (n = 40; P < 0.05). CONCLUSION This study has confirmed that the expression of MMP-1, MMP-2, TIMP-1, and TIMP-2 genes is important for the pathogenesis of periapical inflammatory lesions. Since the abovementioned markers were not differentially expressed in periapical granulomas and radicular cysts, the challenge of finding the genetic differences between the two lesions still remains.

21 RRJVS| Volume 2 | Issue 1 | March, 2016 Genetic Diversity of Arabian Horse from Stud "Borike" (Bosnia and Herzegovina) Using Microsatellite Markers Dunja Rukavina1*, Danica Hasanbašić1, Adaleta Durmić-Pašić2, Belma Kalamujić2, Amir Zahirović3, Jasmin Ramić2, Naris Pojskić2 1Department for Biology, Veterinary Faculty, University of Sarajevo, Zmaja od Bosne 90. Bosnia and Herzegovia. 2Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Zmaja od Bosne 8. Bosnia and Herzegovia. 3Department for Internal Diseases, Veterinary Faculty, University of Sarajevo, Zmaja od Bosne 90, Bosnia and Herzegovia.

The genetic structure of Alpine salamander (Salamandra atra, Laurenti 1768) populations in the Dinarides with respect to continuous populations in the Alps is still poorly understood. To compare Dinaric populations with the nearest Alpine populations, eleven populations were genotyped using seven microsatellite loci. Two major groups were detected: a more diverse Alpine group in the Steiner and Julian Alps, and a less diverse Dinaric group. The Pokljuka population was assigned to the Dinaric group despite its geographical location in the Alps, placing the divide between major groups north of the southern Alpine orographic boundary. Bottlenecks dated at the end of the last glaciation event were suggested for Alpine populations, but not for Dinaric populations. Genetic signatures of migration were detected within Dinaric and Alpine regions, but not between them. Populations from the Prenj Mountain (Bosnia-Herzegovina), where the subspecies S. atra prenjensis was described, were not genetically distinct from other Dinaric populations. These results suggest that, if the taxon remains valid, S. atra prenjensis should include the entire Dinarides as well as the Pokljuka population located in the Alps.

This paper presents the first estimation of polymorphism of the Bosnian-Herzegovinian-Croatian Shepherd dog Tornjak in Bosnia and Herzegovina using 10 microsatellite loci, which are an integral part of StockMarks® for Canine Genotyping Kit (Applied Biosystems, Foster City, CA, USA). Ten microsatellite loci used in this study are appropriate for assessing the genetic diversity for this breed. Measures of genetic diversity were estimated based on allelic and genotypic calculations, observed (HO) and expected (HE) heterozygosities, deviations from Hardy-Weinberg equilibrium and polymorphism information content (PIC). The lowest genetic diversity was estimated for locus PEZ20, and the highest for PEZ6 locus. Observed and expected mean heterozygosities were 0.7261 and 0.7392, respectively. Statistically significant deviation (p<0.05) from Hardy-Weinberg equilibrium was found for PEZ1, PEZ12, PEZ3 and PEZ6 loci. The PIC values suggested that all markers (100%) are very informative (PIC > 0.5) in terms of their suitability for genetic diversity studies. When all observed parameters are taken into account (observed and expected heterozygosities, PIC, number of detective and effective alleles, number of detected and maximum possible genotypes, major allele frequency and major allele frequency index), we can conclude that PEZ6 locus shows the highest genetic diversity while PEZ3 displays the lowest. However, assuming values of observed and expected heterozygosities, as well as PIC, we consider loci PEZ20 to be the least diverse, but this locus has more effective alleles and more genotypes present than PEZ3. These preliminary results are the first genetic diversity survey of the Bosnian-Herzegovinian-Croatian Shepherd dog Tornjak in Bosnia and Herzegovina and could be useful to the dog breeders in designing and managing breeding strategies. Summarizing the information above, we can conclude that the population of the Bosnian-Herzegovinian-Croatian Shepherd dog Tornjak from B&H is not affected by substantial loss of genetic diversity. Results of our study indicate presence of reasonably high level of genetic variability and lead to a better understanding of this dog breed.

In the present study, for the first time we investigated the genetic diversity among horse breeds from Bosnia and Herzegovina: potential Bosnian and Herzegovinian mountain horse, Arabian horse, Thoroughbred horse and crossbreeds, with special emphasis on the gene pool of potential Bosnian and Herzegovinian mountain horse. In total, 138 animals were genotyped for 17 microsatellite loci. Compared to the other breeds, potential Bosnian and Herzegovinian mountain horse showed quite a high genetic variability. The mean number of alleles was 14.1765. The average observed heterozygosity was 0.6589 and the expected heterozygosity was 0.8451. The mean value of polymorphic information content was 0.8286. The results of AMOVA test showed 8,44% of genetic variation among populations. The highest genetic variation within population was showed by potential Bosnian and Herzegovinian mountain horse (27.13). The same breed showed the highest individual variation (17.35). Overall FST value showed high level of the genetic differentiation among breeds (8.87 %), and the pairwise FST values were all significant. Highest inter-group genetic differentiation was observed among Arabian horse and Thoroughbred horse (groups of pure breeds) and potential Bosnian and Herzegovinian mountain horse. The results show that the potential Bosnian and Herzegovinian mountain horse has a high in-breed variability, more than could be expected. In the gene pool of potential Bosnian and Herzegovinian mountain horse, a part of the gene pool of other breeds is present. Also, these results show that there are very good preconditions for the revitalization of the gene pool of potential Bosnian and Herzegovinian mountain horse. Keywords: Horse, genetic diversity, microsatellites

Objectives: This study investigated the correlation between Tannerella forsythia, Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans at dual sites in concurrent endodontic-periodontal diseases. Material and methods: Samples were collected from endodontium and periodontium in cases of concurrent endodontic-periodontal diseases from thirty participants. The sensitivity and specificity of SYBR Green real-time PCR was used to identify the targeted species. Absolute number of targeted genome copies in tested samples were extrapolated from respective calibration curve. Results: No statistical difference was found in the number of detected endodonticperiodontal pathogens between the endodontium and periodontium. The Pearson test detected significant correlation (P<0.001) between targeted bacteria; T. forsythia, F. nucleatum, and P. gingivalis from endodontic-periodontal lesions. Synergistic component observed separately in endodontic biofilm was found only between T. forsythia and F. nucleatum (r=0.380, P=0.03) while in periodontal biofilm T. forsythia, F. nucleatum and P. gingivalis gave high synergism result (P<0.0001). Correlation analysis showed that T. forsythia in primary endodontic infection and in periodontal lesion was significantly decreased with the increase of patients age (r=-0.308, P=0.017). Conclusions: Correlation between targeted bacterial species levels from concurrent endodontic-periodontal diseases confirmed that coronal and cervical dentinal tubules may represent a viable pathway that allows spreading and maintaining of dual sites infection. Periodontal bacteria detected in root canal of concurrent endodonticperiodontal infections may originate from the local periodontal lesions.

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.

Being a crossroad of many ancient and recent historical migrations, Bosnia and Herzegovina (B&H) represents unique spot of multicultural and social diversity. The main aim of this study was to assess genetic structure of three local populations of mountain area from central part of B&H using mtDNA HVS-1 as an informative marker for population genetics studies. A 444 bp HVS-1 segment of control region of mtDNA extracted from buccal swabs was PCR amplified and sequenced. Haplotype and nucleotide diversity, average number of nucleotide differences, AMOVA and pairwise FST based on mtDNA haplotype and haplogroup frequencies were calculated. NJ tree was constructed based on pairwise FST results. Tajima’s D was calculated to evaluate population demographic status.

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.

Background: From a demographic and genetic perspective, Bosnia and Herzegovina is interwoven with a number of differentially isolated local populations of indigenous people with different population and religious backgrounds. Aim: In order to estimate their genetic structure, this study investigated the frequencies of 10 Alu polymorphic loci in 10 regional populations distributed across Bosnia and Herzegovina. Genetic differentiation among the three major population groups in Bosnia and Herzegovina was estimated. Subjects and methods: DNA from 506 unrelated individuals was extracted from buccal swabs using the salting-out extraction method. Each DNA sample was PCR-amplified using locus-specific primers. Results: Gene diversity values showed similarity in all analysed populations and ranged from 0.305–0.328. FST values for all loci showed that most variability is found within populations. Overall FST for all loci and AMOVA indicated that most variability was detected within populations. Conclusion: Results of this study are in agreement with the previous studies, indicating that the three populations in Bosnia and Herzegovina have the same genetic background. There is no significant differentiation among regional populations, pointing to absence of geographic influence. The Bosnian population is clearly located within the European gene pool.

There are two major theories for inheritance of Rh blood group system: Fisher - Race theory and Wiener theory. Aim of this study was identifying frequency of RHDCE alleles in Bosnian - Herzegovinian population and introduction of this method in screening for Rh phenotype in B&H since this type of analysis was not used for blood typing in B&H before. Rh blood group was typed by Polymerase Chain Reaction, using the protocols and primers previously established by other authors, then carrying out electrophoresis in 2-3% agarose gel. Percentage of Rh positive individuals in our sample is 84.48%, while the percentage of Rh negative individuals is 15.52%. Inter-rater agreement statistic showed perfect agreement (K=1) between the results of Rh blood system detection based on serological and molecular-genetics methods. In conclusion, molecular - genetic methods are suitable for prenatal genotyping and specific cases while standard serological method is suitable for high-throughput of samples.

The mitochondrial DNA (mtDNA) polymorphisms in Bosnian human population was analyzed by means of hypervariable segment I and II (HVSI and HVSII) sequencing and restriction fragment-length polymorphism analysis of the mtDNA coding region. The results suggest that shaping the genetic structure of recent Bosnian population likely to be affected by the expansion from the European glacial refuges area at the end of the Last Glacial Maximum (LGM), postglacial expansions from southwestern refuges of Europe, the Italian Peninsula and the dispersion in periods of more recent historical events, from the East European Plain. Especially interesting feature of the Neolithic expansion in this area is the ancient African/South Asian haplogroup N1a with the HVSI variant 16147G, which is almost absent in Europe. The haplotyps HVSI with variant 16147G suggest the colonization of the Northeast Bosnia region by Neolithic communities in the Early Neolithic period of expansion through Europe, as evidenced by the archaeological remains of the Starcevo culture.