Josip Crnjac

Analysis of 12 X-chromosomal markers in the population of central Croatia

Investigator® Argus X-12 Kit is a commercially available set that allows simultaneous PCR amplification of 12 X-STR markers belonging to four linkage groups (LG). To assess the forensic efficiency of these markers for the population of central Croatia and consequent applicability in routine forensic casework, DNA from 200 blood samples of unrelated donors (100 female and 100 male) was amplified by Investigator® Argus X-12 Kit and analyzed by capillary electrophoresis. Statistical computations based on allele and haplotype frequencies for LG1 - LG4 were performed using Arlequin 3.5 software and on-line tool available at ChrX-STR.org. In female samples, all X-STR markers were in Hardy-Weinberg equilibrium (HWE). The most informative marker for central Croatia population was DXS10135 with polymorphism information content (PIC) 0.9296. The least polymorphic locus was DXS8378 (PIC=0.6363). Power of discrimination (PD) varied from 0.6968 to 0.9336 in male and from 0.8476 to 0.9916 in female samples. Combined PD exceeded 0.999999999 in both men and women. In male samples, linkage disequilibrium (LD) test revealed significant association (P=0.0000) of one marker pair in LG4 and two marker pairs in LG3. Portion of observed haplotypes in the number of possible haplotypes varied from 2.86% to 7.47% across all LGs. LG1 was the most informative with haplotype diversity (H) 0.9972. High PD of all analyzed markers exhibited for central Croatia population confirms suitability of Investigator® Argus X-12 for forensic pertinence. Moreover, results of this study will be included in establishing a national reference X-STR database based on 12 X-STR loci, which is necessary for the correct interpretation of the forensic casework results.

Synthetic vs natural scaffolds for human limbal stem cells

Aim To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. Methods Human placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. Results Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. Conclusion Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purposes.

Analysis of 12 X-STR loci in the population of south Croatia

The aim of the study was to assess forensic pertinence of 12 short tandem repeats (STRs) on X-chromosome in south Croatia population. Investigator® Argus X-12 kit was used to co-amplify 12 STR loci belonging to four linkage groups (LGs) on X-chromosome in 99 male and 98 female DNA samples of unrelated donors. PCR products were analyzed by capillary electrophoresis. Population genetic and forensic parameters were calculated by the Arlequin and POPTREE2 software, and an on-line tool available at ChrX-STR.org. Hardy–Weinberg equilibrium was confirmed for all X-STR markers in female samples. Biallelic patterns at DXS10079 locus were detected in four male samples. Polymorphism information content for the most (DXS10135) and the least (DXS8378) informative markers was 0.9212 and 0.6347, respectively. In both male and female samples, combined power of discrimination exceeded 0.999999999. As confirmed by linkage disequilibrium test, significant association of marker pair DXS10074-DXS10079 (P = 0.0004) within LG2 and marker pair DXS10101-DXS10103 (P = 0.0003) within LG3 was found only in male samples. Number of observed haplotypes in our sample pool amounted 3.01, 7.53, 5 and 3.25% of the number of possible haplotypes for LG1, LG2, LG3 and LG4, respectively. According to haplotype diversity value of 0.9981, LG1 was the most informative. In comparison of south Croatia with 26 world populations, pair-wise

Expanded Croatian 12 X-STR loci database with an overview of anomalous profiles

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Investigator Argus X-12 study on the population of northern Croatia

FST* values increase in parallel with geographical distance. Overall statistical assessment confirmed suitability of Investigator® Argus X-12 kit for forensic casework in both identification and familial testing in the population of south Croatia.

The effects of feed supplemented with Agaricus bisporus on health and performance of fattening broilers.

Abstract Animal hairs are an apt surface for retention of forensic trace epithelial samples. The aim of this study was threefold: to evaluate different methods of sample collection (moistened and dry swabs) and DNA extraction (Chelex® 100 method, Qiagen EZ1® DNA Investigator Kit), as well as to examine the morphological differences of hair fibres between two species (dog, sheep) and their ultimate impact on sample collection and processing. Our preliminary findings suggest that the use of EZ1® DNA Investigator Kit yields donor DNA profiles of higher quality. The results of different sample collection methods have shown intraspecific variations that require further investigation. The ability of retention and subsequent extraction of trace DNA appears to be similar between the two species, despite significant morphological differences between their coat hairs.

Imunofenotipizacija dendritičnih stanica u organiziranim limfnim tkivima probavnog sustava svinja.

In order to implement X-chromosome short tandem repeat (X-STR) typing into routine forensic practice, reference database of a given population should be established. Therefore we extended already published data with additional 397 blood samples from unrelated Croatian citizens, and analyzed the total of 995 samples (549 male and 446 female) typed by Investigator® Argus X-12 Kit. To test genetic homogeneity of consecutively processed five historic-cultural regions covering the entire national territory, we calculated pairwise Fst genetic distances between regions based on allele and full haplotype frequencies. Since the comparison did not yield any statistically significant difference, we integrated STR profile information from all regions and used the whole data set to calculate forensic parameters. The most informative marker is DXS10135 (polymorphism information content (PIC = 0.929) and the most informative linkage group (LG) is LG1 (PIC = 0.996). We confirmed linkage disequilibrium (LD) for seven marker pairs belonging to LG2, LG3 and LG4. By including LD information, we calculated cumulative power of discrimination that amounted to 0.999999999997 in females and 0.999999005 in males. We also compared Croatia with 13 European populations based on haplotype frequencies and detected no statistically significant Fst values after Bonferroni correction in any LG. Multi-dimensional scaling plot revealed tight grouping of four Croatian regions amongst populations of southern, central and northern Europe, with the exception of northern Croatia. In this study we gave the first extensive overview of aberrant profiles encountered during Investigator® Argus X-12 typing. We found ten profiles consistent with single locus duplication followed by tetranucleotide tract length polymorphism. Locus DXS10079 is by far the most frequently affected one, presumably mutated in eight samples. We also found four profiles consistent with X-chromosome aneuploidy (three profiles with XXX pattern and one profile with XXY pattern). In conclusion, we established integral forensic Croatian X-chromosome database, proved forensic pertinence of Investigator® Argus X-12 Kit for the entire Croatian population and identified locus DXS10079 as a potential duplication hotspot.