Gordan Mršić

Improving the controlled delivery formulations of caffeine in alginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium

Alginate-based blends consisting of carrageenan, pectin, chitosan or psyllium husk powder were prepared for assessment of the best formulation aimed at encapsulation of caffeine. Alginate-pectin blend exhibited the lowest viscosity and provided the smallest beads. Alginate-psyllium husk blend was characterised with higher viscosity, yielding the largest bead size and the highest caffeine encapsulation efficiency (83.6%). The release kinetics of caffeine indicated that the porosity of alginate hydrogel was not reduced sufficiently to retard the diffusion of caffeine from the beads. Chitosan coated alginate beads provided the most retarded release of caffeine in water. Morphological characteristics of beads encapsulating caffeine were adversely affected by freeze drying. Bitterness intensity of caffeine-containing beads in water was the lowest for alginate-psyllium beads and chitosan coated alginate beads. Higher sodium alginate concentration (3%) for production of hydrogel beads in combination with psyllium or chitosan coating would present the most favourable carrier systems for immobilization of caffeine.

Flow Properties of Commonly Used Food Powders and Their Mixtures

The aim of this study was to get an insight on flow properties of five different food powders and their mixtures frequently used in industrial production of foodstuffs and in households. The studied food powders were flour, maize semolina, sugar, cocoa, and skim milk powder together with their mixtures. Based on cohesion index, samples were categorized as follows: easy flowing—sugar powder, skim milk powder, maize semolina, and mixture 3 (wheat flour and sugar powder); cohesive—flour and mixture 1 (wheat flour and maize semolina); very cohesive—mixture 4 (wheat flour, cocoa, and sugar powder), and extremely cohesive—cocoa powder and mixture 2 (cocoa, sugar, and skim milk powder). Based on powder flow speed dependency test, positive multiple correlations were found between four powder mixtures and their basic components at all speeds. Flour, maize semolina, and mixtures 1 and 3 did not show any susceptibility to caking, while sugar, skim milk powder, cocoa powder, and mixtures 2 and 4 showed an increasing cake height ratio indicating their susceptibility to caking.

Morphogenesis, volume and number of hop (Humulus lupulus L.) glandular trichomes, and their influence on alpha-acid accumulation in fresh bracts of hop cones

Morphogenesis, volume and number of hop (Humulus lupulus L.) glandular trichomes, and their influence on alpha-acid accumulation in fresh bracts of hop cones The ESEM investigations revealed the morphogenesis of peltate glandular trichomes, which was divided into five phases. In phase one, new peltate glandular trichomes were initiated; in phase two, they were differentiated; in phase three trichomes grew vigorously; in phase four they were determined; in the fifth and final phase they came to maturity. Volume of glandular trichome during the different phases of morphogenesis varied from 0.25 × 10-2 mm3 in phase 1, to 1.95 × 10-2 mm3 in phase 5. More glandular trichomes are placed on the base of the adaxial side of bracts (average 7 mm-2) than on the base of abaxial side (average 5.8 mm-2). In this research, positive Spearmans rank order correlations were found between the average number of glandular trichomes and content of α-acids as well as between the average volume of glandular trichomes and content of α-acids.

Analysis of 8 X-chromosomal markers in the population of central Croatia

Aim To analyze 8 X-linked short tandem repeat (STR) markers in the population of central Croatia and to evaluate their forensic efficiency. Methods We carried out a statistical analysis of the data from previously performed genetic analyses, collected during routine forensic work by the Forensic Science Centre ‘‘Ivan Vučetić.’’ Mentype® Argus X-8 PCR amplification kit was used for typing the data of 99 unrelated healthy women and 78 men from central Croatia. Haplotype frequencies were calculated only in male samples. Arlequin 3.5 software was used to assess Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), observed and expected heterozygosity. Power of discrimination (PD) for men and women, polymorphism information content (PIC), power of exclusion, and mean exclusion chance for deficiency cases, normal trios, and duos were determined using online database ChrX-STR.org. Results In female samples, deviations from HWE (P = 0.006) for each locus were not found. LD test performed both on female and male samples revealed no significant association between markers (P = 0.002). DXS10135 was the most polymorphic locus (PIC = 0.931). PD varied from 0.692 to 0.935 in male and from 0.845 to 0.992 in female samples. Combined PD reached 99.999999% in men and 99.9999999999% in women. Conclusion Performed analyses revealed that the studied marker set contained polymorphic markers with high power of discrimination. We can conclude that Mentype® Argus X-8 PCR kit is suitable for application in the population of central Croatia. Results of this study, together with collected allele and haplotype frequencies, are the first step in establishing a national reference X-STR database based on 8 X-STR loci.

Defining centric diatoms of most relevant phytoplankton functional groups in deep karst lakes

Phytoplankton in karst lakes is characterized by co-occurrence of chrysophytes (Chrysophyceae), dinoflagellates (Dinophyceae), and diatoms (Bacillariophyta) as the most diverse and abundant group. Using Reynolds functional approach in ecological phytoplankton investigations makes karstic lakes comparable and facilitates interpretation of their responses to changing environmental conditions. Accurate taxonomic identification to species level, based on precise resolution of specific characteristics by electron microscopy, is essential in order to correctly associate species into coda. This paper defines the most abundant centric diatoms and coexisting species in the phytoplankton of karst lakes in Croatia as a contribution to better description of phytoplankton functional groups dominated by centric diatoms. Several representatives for diatom-based Reynolds functional groups of four lakes in Croatia were described in this paper: Pantocsekiella costei and Stephanocostis chantaica for group A, Pantocsekiella ocellata and Cyclotella distinguenda for group B, and Stephanodiscus neoastraea for group C, together with their ecological preferences which clearly correspond to pertaining functional classification. Selected and coexisting functional groups define natural, oligo- to mesotrophic karst deep lake systems.

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.

Effect of ultraviolet C radiation on biological samples

Aim To examine the influence of ultraviolet C (UVC) radiation on blood, saliva, semen, and naked DNA samples for preventing DNA cross-contamination on working surfaces in laboratories. Methods Blood, saliva, semen, and DNA isolated from buccal swab samples were obtained from a single male donor and applied to the laboratory working surfaces. UVC radiation was applied to these diluted and undiluted samples with or without previous decontamination of the working surfaces with 10% sodium hypochlorite and 20% ethanol. Genomic DNA was extracted using Chelex. After quantification, DNA was amplified using the AmpFlSTR® NGM™ PCR Amplification Kit. We tested and statistically analyzed DNA concentration, UVC dose, sample volume, radiation time, the number of correctly detected alleles on genetic loci, and the number of correctly detected alleles in four groups in which 16 loci were divided. Results When working surfaces were not decontaminated and were treated only with UVC radiation in the laboratory, the genetic profile for naked DNA could not be obtained after 2 minutes of UVC radiation and for saliva after 54 hours. For blood and semen, a partial genetic profile was obtained even after 250 hours of UVC radiation in the laminar. When working surfaces were decontaminated with 10% sodium hypochlorite and 20% ethanol, genetic profile could not be obtained for naked DNA after 2 minutes, for saliva after 4 hours, for blood after 16 hours, and for semen after 8 hours of UVC radiation in the laboratory. Conclusion It is recommended to carefully and thoroughly clean working surfaces with 10% sodium hypochlorite and 20% ethanol followed by minimal 16-hour UVC exposure (dose approximately 4380 mJ/cm2) for complete and successful decontamination.

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