Institute of Molecular Genetics and Genetic Engineering

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Alginate is a natural polymer of marine origin and, due to its exceptional properties, has great importance as an essential component for the preparation of hydrogels and scaffolds for biomedical applications. The design of biologically interactive hydrogels and scaffolds with advanced, expected and required properties are one of the key issues for successful outcomes in the healing of injured tissues. This review paper presents the multifunctional biomedical applications of alginate-based hydrogels and scaffolds in selected areas, highlighting the key effect of alginate and its influence on the essential properties of the selected biomedical applications. The first part covers scientific achievements for alginate in dermal tissue regeneration, drug delivery systems, cancer treatment, and antimicrobials. The second part is dedicated to our scientific results obtained for the research opus of hydrogel materials for scaffolds based on alginate in synergy with different materials (polymers and bioactive agents). Alginate has proved to be an exceptional polymer for combining with other naturally occurring and synthetic polymers, as well as loading bioactive therapeutic agents to achieve dermal, controlled drug delivery, cancer treatment, and antimicrobial purposes. Our research was based on combinations of alginate with gelatin, 2-hydroxyethyl methacrylate, apatite, graphene oxide and iron(III) oxide, as well as curcumin and resveratrol as bioactive agents. Important features of the prepared scaffolds, such as morphology, porosity, absorption capacity, hydrophilicity, mechanical properties, in vitro degradation, and in vitro and in vivo biocompatibility, have shown favorable properties for the aforementioned applications, and alginate has been an important link in achieving these properties. Alginate, as a component of these systems, proved to be an indispensable factor and played an excellent "role" in the optimal adjustment of the tested properties. This study provides valuable data and information for researchers and demonstrates the importance of the role of alginate as a biomaterial in the design of hydrogels and scaffolds that are powerful medical "tools" for biomedical applications.

Eight 1,3,4-oxadiazoles and eight of their diacylhydrazine precursors were synthesized and examined for their antioxidative potential using different tests.

INTRODUCTION: The objective of this study was to characterize the lactobacilli from the human oral cavity as a potential source of probiotic strains. METHODS: Samples were collected from four different locations within the oral cavity: surface of healthy tooth, oral mucous membrane, surface of tooth decay and deep tooth decay. On the basis of morphological and biochemical properties eight categories were formed and 26 isolates were selected for further characterization. The isolates were determined as Lactobacillus sp. using primers specific for 16S rDNA. Sequencing of 16S rDNA genes and repetitive sequence-based polymerase chain reactions were used for determination to species and subspecies levels. RESULTS: Predominant species were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus salivarius and Lactobacillus paracasei subsp. paracasei, while Lactobacillus acidophilus, Lactobacillus cellobiosus, Lactobacillus delbrueckii subsp. lactis and Lactobacillus gasseri were also present. The isolates Lactobacillus salivarius BGHO1, Lactobacillus fermentum BGHO36 and BGHO64, Lactobacillus gasseri BGHO89 and Lactobacillus delbrueckii subsp. lactis BGHO99 exhibited antagonistic action on the growth of Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, Salmonella enteritidis, Streptococcus pneumoniae and Streptococcus mutans, but not on growth of Candida albicans. Moreover, the isolates L. salivarius BGHO1 and L. gasseri BGHO89 were tolerant to low pH and high concentration of bile salts. CONCLUSION: Taken together, these findings imply that L. salivarius BGHO1 and L. gasseri BGHO89 might be subjects for additional investigation as potential probiotic strains.

BACKGROUND: The cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes. METHODS: Patients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed. RESULTS: Variants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01<Praw<0.0001 at IL1B, TLR9, TNFα, CD95, STAT3 and TNFR). The intragenic background of F508del-CFTR chromosomes determined disease severity and manifestation of the basic defect (Praw=0.0009). Allele distributions comparing transmitted and non-transmitted alleles were distorted at several loci unlinked to CFTR. CONCLUSIONS: The inherited capabilities of the innate and adaptive immune system determine the manifestation of the CF basic defect. Variants on F508del-CFTR chromosomes contribute to the observed patient-to-patient variability among F508del-CFTR homozygotes. A survivor effect, manifesting as a transmission disequilibrium at many loci, is consistent with the improvement of clinical care over the last decades, resulting in a depletion of risk alleles at modifier genes. Awareness of non-genetic factors such as improvement of patient care over time is crucial for the interpretation of CF modifier studies.

A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized. The prtR gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its alpha(S1)- and beta-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L. rhamnosus strains tested. Proteinase extracts of the BGT10 strain obtained with Ca(2+)-free buffer at pH 6.5 were proteolytically active. The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions. The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.

A series of 16 Pt(<sc>ii</sc>) complexes with 8-hydroxyquinolines and sulfoxide/phosphine ligands were synthetized, characterized and evaluated for cytotoxic and embryotoxic activity.

N This study confirms an association between MBL2 variants and the cystic fibrosis pulmonary phenotype, and therefore it is very likely that mannose binding lectin protein is indeed a modulating factor in cystic fibrosis disease.

Various constituents of the olive tree (Olea europaea) have been traditionally used in the treatment of infection, inflammation, prevention of chronic diseases, cardiovascular disorders and cancer. The anticancer potential of dry olive leaf extract (DOLE) represents the net effect of multilevel interactions between different biologically active compounds from the extract, cancer cells and conventional therapy. In this context, it was of primary interest to evaluate the influence of DOLE on progression of the highly malignant, immuno- and chemoresistant type of skin cancer-melanoma. DOLE significantly inhibited proliferation and subsequently restricted clonogenicity of the B16 mouse melanoma cell line in vitro. Moreover, late phase tumor treatment with DOLE significantly reduced tumor volume in a syngeneic strain of mice. DOLE-treated B16 cells were blocked in the G(0) /G(1) phase of the cell cycle, underwent early apoptosis and died by late necrosis. At the molecular level, the dying process started as caspase dependent, but finalized as caspase independent. In concordance, overexpression of antiapoptotic members of the Bcl-2 family, Bcl-2 and Bcl-XL, and diminished expression of their natural antagonists, Bim and p53, were observed. Despite molecular suppression of the proapoptotic process, DOLE successfully promoted cell death mainly through disruption of cell membrane integrity and late caspase-independent fragmentation of genetic material. Taken together, the results of this study indicate that DOLE possesses strong antimelanoma potential. When DOLE was applied in combination with different chemotherapeutics, various outcomes, including synergy and antagonism, were observed. This requires caution in the use of the extract as a supplementary antitumor therapeutic.

CONTEXT: Alpha-1-antitrypsin (A1AT) is the most abundant liver-derived, highly polymorphic, glycoprotein in plasma. Hereditary deficiency of alpha-1-antitrypsin in plasma (A1ATD) is a consequence of accumulation of polymers of A1AT mutants in endoplasmic reticulum of hepatocytes and other A1AT-producing cells. One of the clinical manifestations of A1ATD is liver disease in childhood and cirrhosis and/or hepatocellular carcinoma (HCC) in adulthood. Epidemiology and pathophysiology of liver failure in early childhood caused by A1ATD are well known, but the association with hepatocellular carcinoma is not clarified. The aim of this article is to review different aspects of association between A1AT variants and hepatocellular carcinoma, with emphasis on the epidemiology and molecular pathogenesis. The significance of A1AT as a biomarker in the diagnosis of HCC is also discussed. EVIDENCE ACQUISITIONS: Search for relevant articles were performed through Pub Med, HighWire, and Science Direct using the keywords "alpha-1-antitrypsin", "liver diseases", "hepatocellular carcinoma", "SERPINA1". Articles published until 2011 were reviewed. RESULTS: Epidemiology studies revealed that severe A1ATD is a significant risk factor for cirrhosis and HCC unrelated to the presence of HBV or HCV infections. However, predisposition to HCC in moderate A1ATD is rare, and probably happens in combination with HBV and/or HCV infections or other unknown risk factors. It is assumed that accumulation of polymers of A1ATD variants in endoplasmic reticulum of hepatocytes leads to damage of hepatocytes by gain-of-function mechanism. Also, increased level of A1AT was recognized as diagnostic and prognostic marker of HCC. CONCLUSIONS: Clarification of a carcinogenic role for A1ATD and identification of proinflammatory or some still unknown factors that lead to increased susceptibility to HCC associated with A1ATD may contribute to a better understanding of hepatic carcinogenesis and to the development of new drugs.

Pseudomonas sp. ATCC19151 harbors a gene encoding a putative alkylsulfatase (sdsA). Here we report a growth ability of this strain in minimal media containing 0.5, 0.75, and 1% sodium dodecyl sulfate as the sole carbon source. The most prominent growth was detected for the minimal medium with 0.5% SDS, so this concentration of SDS was used to monitor Pseudomonas sp. ATCC19151 SDS biodegradation dynamics. Bacterial growth coincided with the disappearance of SDS. Antibiotic susceptibility was tested as well. Pseudomonas sp. ATCC19151 was resistant to six out of nine tested antibiotics, including ampicillin, tetracycline, chloramphenicol, tobramycin, nalidixic acid, and gentamycin.

Using cultivation-dependant method, we isolated 184 strains from fresh and old bee bread, pollen, larvae and adults of solitary bee Osmia cornuta. The 16S rDNA sequencing of 79 selected isolates gave the final species-specific identification of strains. Phylogenetic analysis indicated that microbiota isolated from five different sources were represented with 29 species within three different phyla, Firmicutes with 25 species, Actinobacteria with only one species and Proteobacteria with three species of Enterobacteriaceae. Bacterial biodiversity presented with Shannon-Wiener index (H') was highest in the alimentary tract of adults and old bee bread (H' = 2.43 and H' = 2.53, respectively) and in the same time no dominance of any species was scored. On the contrary, results obtained for Simpson index (D) showed that in pollen samples the dominant species was Pantoea agglomerans (D = 0.42) while in fresh bee bread that was Staphylococcus sp. (D = 0.27). We assume that microbial diversity detected in the tested samples of solitary bee O. cornuta probably come from environment.

In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.

BACKGROUND: Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients. METHODS: We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results. RESULTS: Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed. CONCLUSIONS: High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates.

Molecular modifications on sulfathiazole to overcome sulfonamide resistance: the discovery of selective antibacterial agents against <italic>Staphylococcus aureus</italic>.

INTRODUCTION: Lactobacillus salivarius BGHO1, a human oral isolate with antagonistic activity against growth of Streptococcus mutans, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, and Salmonella enteritidis, probably produces more than one proteinaceous antimicrobial substance. The objective of this study was the purification of a bacteriocin, named LS1, produced by L. salivarius BGHO1. METHODS: A simple and fast procedure for bacteriocin purification was developed, consisting of reverse-phase chromatography of the ammonium sulfate precipitate of cell-free culture supernatant by fast protein liquid chromatography and high-performance liquid chromatography, followed by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with the subsequent extraction of bacteriocin from the gel. RESULTS: The supernatant of L. salivarius BGHO1 culture retained its antimicrobial activity after boiling in a water bath for 15 min. Its antimicrobial activity was also maintained even after treatment for 20 min at 121 degrees C in an autoclave. Bacteriocin LS1 was purified to homogeneity. The molecular mass of bacteriocin LS1 was estimated to be approximately 10 kDa, based on tricine SDS-PAGE. During purification, another compound with antimicrobial activity, produced by L. salivarius BGHO1, was detected. The molecular mass of this compound was estimated to be approximately 5 kDa, based on tricine SDS-PAGE. CONCLUSION: Our results imply that LS1 is most probably a new bacteriocin, different from previously described bacteriocins produced by L. salivarius strains. The purification of bacteriocin LS1 enabled the further characterization of LS1 on both the molecular and genetic levels.

The PsrA transcriptional regulator is involved in stationary phase induced transcriptional regulation of rpoS and in negative auto-regulation in Pseudomonas aeruginosa. This study was designed to determine whether other loci were regulated by PsrA in P. aeruginosa. Computer search was performed of the PsrA binding motif (G/CAAAC N(2-4) GTTTG/C) against the P. aeruginosa genome sequence. Four of 14 analysed promoters responded to and bound PsrA; (i) divergent promoters controlling PA2952/PA2951 and PA2953, (ii) promoter of PA0506 and (iii) upstream region of PA3571. Promoters PA0506 and PA2952-PA2951 were regulated negatively whereas promoters of PA2953 and PA3571 were regulated positively by PsrA. Two dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D SDS-PAGE) analysis on total proteins from P. aeruginosa PAO1 and psrA knock-out derivative was also performed resulting in the identification of 11 protein spots which were differentially regulated. These studies have indicated PsrA as a global regulator.

Many species of Prunus display an S-RNase-based gametophytic self-incompatibility (SI), controlled by a single highly polymorphic multigene complex termed the S-locus. This comprises tightly linked stylar- and pollen-expressed genes that determine the specificity of the SI response. We investigated SI of Prunus tenella, a wild species found in small, isolated populations on the Balkan peninsula, initially by pollination experiments and identifying stylar-expressed RNase alleles. Nine P. tenella S-RNase alleles (S(1)-S(9)) were cloned; their sequence analysis showed a very high ratio of non-synonymous to synonymous nucleotide substitutions (K(a)/K(s)) and revealed that S-RNase alleles of P. tenella, unlike those of Prunus dulcis, show positive selection in all regions except the conserved regions and that between C2 and RHV. Remarkably, S(8)-RNase, was found to be identical to S(1)-RNase from Prunus avium, a species that does not interbreed with P. tenella and, except for just one amino acid, to S(11) of P. dulcis. However, the corresponding introns and S-RNase-SFB intergenic regions showed considerable differences. Moreover, protein sequences of the pollen-expressed SFB alleles were not identical, harbouring 12 amino-acid replacements between those of P. tenella SFB(8) and P. avium SFB(1). Implications of this finding for hypotheses about the evolution of new S-specificities are discussed.

Combination therapy may be applied in the case of gold(<sc>iii</sc>) and silver(<sc>i</sc>) complexes with aromatic nitrogen-containing heterocycles to improve their antimicrobial activity and reduce toxic-side effects.