Aleksandar Vojta - Academia.edu (original) (raw)

Papers by Aleksandar Vojta

Clinical Cancer Research, 2016

Alternative glycosylation has significant structural and functional consequences on immunoglobuli... more Alternative glycosylation has significant structural and functional consequences on immunoglobulin G and consequently also cancer immunosurveilance. Due to technological limitations, the effects of highly heritable individual variations and the differences in the dynamics of changes in IgG glycosylation on colorectal cancer (CRC) were never investigated before. Using recently developed high-throughput UPLC technology for IgG glycosylation analysis we analysed IgG glycome composition in 760 CRC patients and 538 matching controls. Effects of surgery were evaluated in 28 patients sampled before and three times after surgery. A predictive model was built using regularized logistic regression and evaluated using a 10-cross validation procedure. Furthermore, IgG glycome composition was analysed in 39 plasma samples collected before initial diagnosis of CRC. We have found that CRC associates with decrease in IgG galactosylation, IgG sialylation and increase in core-fucosylation of neutral glycans with concurrent decrease of core fucosylation of sialylated glycans. While a model based on age and sex did not show discriminative power (AUC=0.499), the addition of glycan variables into the model considerably increased the discriminative power of the model (AUC=0.755). However, none of these differences were significant in the small set of samples collected before the initial diagnosis. Considering the functional relevance of IgG glycosylation for both tumour immunosurveilance and clinical efficacy of therapy with monoclonal antibodies, individual variation in IgG glycosylation may turn out to be important for prediction of disease course or the choice of therapy, thus warranting further, more detailed studies of IgG glycosylation in CRC.

Small Ruminant Research, 2013

"Hematological and biochemical tests are widely used for assessment of health status and dia... more "Hematological and biochemical tests are widely used for assessment of health status and diagnosis of disease in domestic animals. They are almost indispensible in health management of organic flocks, but environmental conditions and food supply can be highly variable in organic farming, which may cause levels of many analytes to lie outside the published reference intervals. We investigated key hematological and biochemical parameters in organically raised sheep in Croatia. Different (mostly indigenous) breeds raised in various geographic regions were evaluated throughout the year. We identified key parameters that varied under different conditions using classification based on machine learning. Our findings suggest that location (which determines climate and food availability) has a profound effect on hematological and biochemical values in organically raised sheep. The effect of location was more pronounced than that of breed and season. The results clearly show the merit of establishing specific reference intervals for each flock, especially in organic farming. We demonstrate feasibility of this approach by successful calculation of reference intervals from a small sample (30 individuals) using robust statistical methods. Keywords Organic farming Indigenous breeds;Reference intervals;n Robust statistics"

Scientific reports, 2016

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless,... more Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that t...

Nucleic Acids Research, 2016

Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pat... more Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

Croatica Chemica Acta, Nov 15, 2008

Biochimica et Biophysica Acta (BBA) - General Subjects, 2016

Most eukaryotic proteins are modified by covalent addition of glycan molecules that considerably ... more Most eukaryotic proteins are modified by covalent addition of glycan molecules that considerably influence their function. Aberrant glycosylation is profoundly involved in malignant transformation, tumor progression and metastasis. Some glycan structures are tumor-specific and reflect disturbed glycan biosynthesis pathways. We analyzed DNA methylation and expression of 86 glyco-genes in melanoma, hepatocellular, breast and cervical cancers using data from publicly available databases. We also analyzed methylation datasets without the available matching expression data for glyco-genes in lung cancer, and progression of melanoma into lymph node and brain metastases. Ten glyco-genes (GALNT3, GALNT6, GALNT7, GALNT14, MGAT3, MAN1A1, MAN1C1, ST3GAL2, ST6GAL1, ST8SIA3) showing changes in both methylation and expression in the same type of cancer belong to GalNAc transferases, GlcNAc transferases, mannosidases and sialyltransferases, which is in line with changes in glycan structures already reported in the same type of tumors. Some of those genes were additionally identified as potentially valuable for disease prognosis. The MGAT5B gene, so far identified as specifically expressed in brain, emerged as a novel candidate gene that is epigenetically dysregulated in different cancers other than brain cancer. We also report for the first time aberrant expression of the GALNT and MAN genes in cancer by aberrant promoter methylation. Aberrant expression of glyco-genes due to aberrant promoter methylation could be a way leading to characteristic glycosylation profiles commonly described in cancer. Methylation status in promoters of candidate glyco-genes might serve as prognostic markers for specific tumors and point to potential novel targets for epigenetic drugs. This article is part of a Special Issue entitled Glycans in personalized medicine, edited by Professor Gordan Lauc.

BioMed research international, 2013

Adaptive response to stress is a fundamental property of living systems. At the cellular level, m... more Adaptive response to stress is a fundamental property of living systems. At the cellular level, many different types of stress elicit an essentially limited repertoire of adaptive responses. Epigenetic changes are the main mechanism for medium- to long-term adaptation to accumulated (intense, long-term, or repeated) stress. We propose the adaptive deregulation of the epigenome in response to stress (ADERS) hypothesis which assumes that the unspecific adaptive stress response grows stronger with the increasing stress level, epigenetically activating response gene clusters while progressively deregulating other cellular processes. The balance between the unspecific adaptive response and the general epigenetic deregulation is critical because a strong response can lead to pathology, particularly to malignant transformation. The main idea of our hypothesis is the continuum traversed by a cell subjected to accumulated stress, which lies between an unspecific adaptive response and patholo...

PLoS ONE, 2014

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are ... more Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-29-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/ nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.

The EMBO Journal, 2004

The Toc core complex consists of the pore-forming Toc75 and the GTPases Toc159 and Toc34. We conf... more The Toc core complex consists of the pore-forming Toc75 and the GTPases Toc159 and Toc34. We confirm that the receptor form of Toc159 is integrated into the membrane. The association of Toc34 to Toc75/Toc159 is GTP dependent and enhanced by preprotein interaction. The N-terminal half of the pSSU transit peptide interacts with high affinity with Toc159, whereas the C-terminal part stimulates its GTP hydrolysis. The phosphorylated C-terminal peptide of pSSU interacts strongly with Toc34 and therefore inhibits binding and translocation of pSSU into Toc proteoliposomes. In contrast, Toc159 recognises only the dephosphorylated forms. The N-terminal part of the pSSU presequence does not influence binding to the Toc complex, but is able to block import into proteoliposomes through its interaction with Toc159. We developed a model of differential presequence recognition by Toc34 and Toc159.

Planta, 2004

Voltage-dependent anion channels (VDACs) are porin-type beta-barrel diffusion pores. They are pro... more Voltage-dependent anion channels (VDACs) are porin-type beta-barrel diffusion pores. They are prominent in the outer membrane of mitochondria and facilitate metabolite exchange between the organelle and the cytosol. Here we studied the subcellular distribution of a plant VDAC-like protein between plastids and mitochondria in green and non-green tissue. Using in vitro studies of dual-import into mitochondria and chloroplasts as well as transient expression of fluorescence-labeled polypeptides, it could be clearly demonstrated that this VDAC isoform targets exclusively to mitochondria and not to plastids. Our results support the idea that plastids evolved a concept of solute exchange with the cytosol different from that of mitochondria.

Journal of Comparative Pathology, 2010

Introduction: Neurological symptoms are rarely caused by neoplasms involving the spinal cord in h... more Introduction: Neurological symptoms are rarely caused by neoplasms involving the spinal cord in horses. Haemangiosarcomas occur most frequently in dogs, but have been infrequently reported in horses. Only a few studies report vertebral haemangiosarcomas in horses. Materials and Methods: An 18-year-old Dutch warmblood gelding with spinal ataxia was humanely destroyed. Necropsy examination revealed an epidural haemorrhagic mass at C3, with compression of the spinal cord and osteolysis of C3 and C4. Thorough examination of other organs revealed no other lesions. The mass was fixed and analyzed by histology and immunohistochemistry for expression of von Willebrand factor. Results: Histological examination of the mass at C3 showed cavities filled with erythrocytes and lined by poorly differentiated endothelial cells, which were confirmed immunohistochemically. These cells were pleomorphic with indistinct cell borders. The nuclei showed anisokaryosis with a prominent nucleolus. Neoplastic endothelial cells invaded the vertebral bodies of C3 and C4 and multifocal osteonecrosis was present.

Journal of Biological Chemistry, 2004

The Toc and Tic translocon facilitate import of preproteins into chloroplasts. In the past, it wa... more The Toc and Tic translocon facilitate import of preproteins into chloroplasts. In the past, it was speculated that several translocon subunits act specifically for different types of precursor proteins or in different tissues. To generate a comprehensive picture of the expression and tissue-specific localization of the translocon subunits, their transcript levels were analyzed in roots and leaves. Certain Tocs and Tics were found to be tissuespecific. The protein composition of the transloci in the envelope membranes of chloroplasts was analyzed to describe the function and possible stoichiometry. In contrast to Tic subunits, several Toc subunits seem to have a high turnover.

Journal of Biological Chemistry, 2007

beta-Barrel-shaped channels of the Omp85 family are involved in the translocation or assembly of ... more beta-Barrel-shaped channels of the Omp85 family are involved in the translocation or assembly of proteins of bacterial, mitochondrial, and plastidic outer membranes. We have compared these proteins to understand the evolutionary development of the translocators. We have demonstrated that the proteins from proteobacteria and mitochondria have a pore diameter that is at least five times smaller than found for the Omp85 in cyanobacteria and plastids. This finding can explain why Omp85 from cyanobacteria (but not the homologous protein from proteobacteria) was remodeled to become the protein translocation pore after endosymbiosis. Further, the pore-forming region of the Omp85 proteins is restricted to the C terminus. Based on a phylogenetic analysis we have shown that the pore-forming domain displays a different evolutionary relationship than the N-terminal domain. In line with this, the affinity of the N-terminal domain to the C-terminal region of the Omp85 from plastids and cyanobacteria differs, even though the N-terminal domain is involved in gating of the pore in both groups. We have further shown that the N-terminal domain of nOmp85 takes part in homo-oligomerization. Thereby, the differences in the phylogeny of the two domains are explained by different functional constraints acting on the regions. The pore-forming domain, however, is further divided into two functional regions, where the distal C terminus itself forms a dimeric pore. Based on functional and phylogenetic analysis, we suggest an evolutionary scenario that explains the origin of the contemporary translocon.

Human Mutation, 2002

This study presents the first molecular data on the basis and the origin of Huntington disease in... more This study presents the first molecular data on the basis and the origin of Huntington disease in Croatia and is the first such analysis performed among a Slavic population. We analyzed three trinucleotide polymorphisms in the HD gene: CAG, CCG and GAG ∆2642 (E2642del) triplets. Analysis of the CAG repeat size among 44 Huntington patients (39-66 CAGs) and 51 normal individuals (9-34 CAGs) showed that the range of the repeats was similar to previous findings. The frequency of the CCG and ∆2642 polymorphic alleles on N and HD chromosomes was found to correlate well with earlier reports for Western European populations. We found significance for both the CCG7 allele (p=0.004) and the ∆2642 allele (p<0.001) among HD chromosomes. The CCG7 allele was overpresented among affected chromosomes (94.6%), but was also the most frequent CCG allele among normal chromosomes (66.7%). Interestingly, the ∆2642 allele was present on 40.5% HD chromosomes compared to only 9.8% of control chromosomes. Our results indicate that HD mutations in Croatia could be of the same origin as in Western populations and also support the multi-step hypothesis for generating new HD alleles. Similar frequencies and distributions of both the CCG and the ∆2642 polymorphisms in Croatia and Western European normal chromosomes indicate that the prevalence rate of HD in Croatia may be as high as in Western populations. Since we estimated a lower prevalence rate (1 : 100,000), we assume that there are still many misdiagnosed and/or unrecognized cases of Huntington disease in Croatia.

FEBS Letters, 2007

The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can b... more The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can be inactivated by phosphorylation in vitro, being arrested in the GDP loaded state. To assess the physiological function of phosphorylation, attoc33 knock out mutants were complemented with a mutated construct mimicking the constitutively phosphorylated state. Our data suggest that the reduced functionality of the mutant protein can be compensated by its upregulation. Chloroplast biogenesis and photosynthetic activity are impaired in the mutants during the early developmental stage, which is consistent with the requirement of atToc33 in young photosynthetic tissues.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2014

Most proteins are glycosylated, with glycans being integral structural and functional components ... more Most proteins are glycosylated, with glycans being integral structural and functional components of a glycoprotein. In contrast to polypeptides, which are fully encoded by the corresponding gene, glycans result from a dynamic interaction between the environment and a network of hundreds of genes. Recent developments in glycomics, genomics and epigenomics are discussed in the context of an evolutionary advantage for higher eukaryotes over microorganisms, conferred by the complexity and adaptability which glycosylation adds to their proteome. Inter-individual variation of glycome composition in human population is large; glycome composition is affected by both genes and environment; epigenetic regulation of &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;glyco-genes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; has been demonstrated; and several mechanisms for transgenerational inheritance of epigenetic marks have been documented. Epigenetic recording of acquired characteristics and their transgenerational inheritance could be important mechanisms used by higher organisms to compete or collaborate with microorganisms.

Analytical Biochemistry, 2005

Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological ... more Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological function. Many protocols exist for this procedure and for the subsequent analysis of proteins. Here, we establish a procedure for preparation and analysis of liposomes with a lipid composition reflecting the outer envelope of chloroplasts. First, the stability of the liposomes in different buffer systems was investigated to provide information for the storage of the reconstituted system. Then, the size of the liposomes created by filtration through a polycarbonate filter dependent on the lipid composition was analyzed. Subsequently, solubilization of the liposomes composed of lipids with the outer envelope composition by dodecylmaltoside and octylglucoside as a preceding step of reconstitution was studied. Finally, we developed a straightforward method to determine the size of liposomes by absorption spectroscopy. The described setup allows the construction of reconstitution protocols, including the final determination of the liposome size.

Acta Veterinaria Hungarica, 2013

Hepatitis E is a viral zoonotic disease infecting swine worldwide. Since pigs represent a likely ... more Hepatitis E is a viral zoonotic disease infecting swine worldwide. Since pigs represent a likely animal reservoir for the hepatitis E virus, the epidemiology of naturally occurring hepatitis E was investigated in Croatian swine herds. Nearly all tested animals were seropositive for antibodies against the hepatitis E virus (55/60, 91.7%). Active infection was detected in all age groups by RT-PCR of viral RNA in serum (8/60, 13.3%) and bile samples (3/37, 8.1%), which was further confirmed by histopathological findings of characteristic lesions in the livers of the infected animals. Three new strains of hepatitis E virus were isolated from Croatian pig herds. Phylogenetic analysis using median-joining networks clustered those Croatian strains with isolates from various parts of the world, indicating their likely origin in international trade. Similarity to human isolates implies a zoonotic potential of Croatian strains, which raises a public health concern, especially in the light of the high prevalence of hepatitis E in the herds studied.