Psycho-social aspects of measures aimed at decreasing prevalence of chronic diseases in the population of returnees in the Osijek Region, Croatia

Recognizing high prevalence of Diabetes mellitus and cardiovascular disorders and low coverage by preventive examinations in the population of returnees, in Osijek Region, we initiated activities for early detection and better managing of chronic diseases. Measures done, were based on public health working methods, such as: education, solidarity and self-responsibility. A special attention was put on psychosocial aspects of the health-related matters. Exactly, free glucose tests were organized with subsequent healthy lifestyles advice session. It was brought to our attention that such intervention should be as much as possible individually oriented, considering many personal and social characteristics of an individual. Gynecological examinations for all women interested in were also organized. On that occasion, women were interviewed in order to get information about their motivation and obstacles for doing preventive examinations and difficulties in adaptation as well. Finally, outcomes of the Project were summarized and put in a larger social context.     

Stomata in some species of genus <Emphasis Type="Italic">Arum</Emphasis> from the Eastern Slavonia and Baranya region

Types and the number of stomata in the following Arum species: Arum italicum Mill., Arum maculatum var. maculatum L. and Arum maculatum var. immaculatum L., Arum alpinum var. pannonicum Terpo. and Arum alpinum var. intermedium Schur. in three different locations in Zablaće, Normanci and Bilje were investigated. The most prevalent stomata type at both upper and lower epidermis for each Arum species was mostly stomata type paracytic, followed by hexacytic, tetracytic or brachyparacytic as far as locality is concerned. Helicocytic type was more prevalent in Arum alpinum var. pannonicum Terpo. and Arum alpinum var. intermedium Schur. at Bilje. Other stomata types were very rare. A striking regularity in the occurrence of stomata types was not found within a single species. Some stomata types, however, were found either at adaxial or abaxial epidermis or were not present at all. The number of stomata per square mm varied from 25 to 651. A statistically significant difference in the number of stomata per square mm at upper and lower epidermis among Arum species was determined in locations Zablaće and Normanci, whereas no statistically significant differences were found in location Bilje.     

Somatic embryogenesis and shoot organogenesis from the hypocotyl slices and free radical scavenging activity of regenerants of collard greens (Brassica oleracea L. var. acephala)

Brassica oleracea var. acephala is an important leafy vegetable that has been widely consumed as a high-nutrient, low-calorie food. Because of the plant’s biennial and self-incompatibility nature, biotechnological approaches are alternative way for propagation and breeding improvements. Since tissue culture studies have been focused in other B. oleracea representatives, the aim of the present study was to achieve effective regeneration protocol distinctive for collard greens, and evaluate the total phenolic content and antioxidant activity of regenerants. The effect of 3 cytokinins [thidiazuron (TDZ), 6-benzyladenine (BA) and 6-furfuryladenine (kinetin, KIN)] at increasing concentrations (0, 5, 7.5, 10, 20 or 30 µM) in combination with tenfold lower concentration of 1-naphtaleneacetic acid (NAA) (0, 0.5, 0.75, 1, 2 or 3 µM, respectively) on the regeneration from hypocotyl slices was studied. Histological analysis revealed the two regeneration pathways, somatic embryogenesis and shoot organogenesis, simultaneously occurred in the same explant, regardless of the cytokinin/NAA combinations used. The regeneration frequency of 95.9%, with 7.5 morphogenic structures regenerated per explant, and the healthy appearance of regenerated plants indicated the optimal combination 20 µM TDZ?+?2 µM NAA. TDZ at 5 µM provided the high somatic embryo proliferation rate by generation of secondary embryos (7.79) along with the lowest rate of their abnormalities. Embryo-developed plants were successfully acclimatised (above 90%). The plants regenerated and proliferated on TDZ-containing media had higher total phenolic content that correlated with the highest free radical scavenging activity (IC₅₀?=?19.09 µg ml^??1).

     

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET¹⁷⁸P), but not rat β-arrestin-1 (PER¹⁷⁷P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK¹⁷⁸P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species.     

Galectin-1 binds mucin in human trophoblast

Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.     

Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.     

Genes for the major structural components of Thermotogales species' togas revealed by proteomic and evolutionary analyses of OmpA and OmpB homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath.     

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.     

Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25–2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.Tamoxifen is an antiestrogenic agent that has been widely and successfully used in the treatment of breast cancer over the past decades (1). Tamoxifen targets and inhibits the estrogen receptor-α, which is expressed in ∼70% of all primary breast tumors and is known to be important in the development and course of the disease. When diagnosed at an early stage, adjuvant systemic tamoxifen therapy can cure ∼10% of the patients (1). In recurrent disease, ∼50% of patients have no benefit from tamoxifen (intrinsic resistance). From the other half of patients who initially respond to therapy with an objective response (OR)¹ or no change (NC), a majority eventually develop progressive disease (PD) due to acquired tamoxifen resistance (2, 3). With the markers available to date we can insufficiently predict therapy response. Therefore, identification of new biomarkers that can more effectively predict response to treatment and that can potentially function as drug targets is a major focus of research.The search for new biomarkers has been enhanced by the introduction of microarray technology. Gene expression studies have resulted in a whole spectrum of profiles for e.g. molecular subtypes, prognosis, and therapy prediction in breast cancer (4–10). Corresponding studies at the protein level are lagging behind because of immature technology. However, protein-level information is crucial for the functional understanding and the ultimate translation of molecular knowledge into clinical practice, and proteomics technologies continue to progress at a rapid pace.Proteomics studies reported so far have mainly been performed with breast cancer cell lines using either two-dimensional gel electrophoresis (11–14) or LC-MS for protein separation (15–17). However, it is known that the proteomic makeup of a cultured cell is rather different from that of a tumor cell surrounded by its native microenvironment (18). Furthermore cell lines lack the required follow-up information for answering important clinical questions. In addition, tumor tissues in general and breast cancer tissues in particular are very heterogeneous in the sense that they harbor many different cell types, such as stroma, normal epithelium, and tumor cells. LCM technology has emerged as an ideal tool for selectively extracting cells of interest from their natural environment (19) and has therefore been an important step forward in the context of genomics and proteomics cancer biomarker discovery research. LCM-derived breast cancer tumor cells have been used for comparative proteomics analyses in the past using both two-dimensional gel electrophoresis (20, 21) and LC-MS (22). This has resulted in the identification of proteins involved in breast cancer prognosis (21) and metastasis (20, 22). Although these studies demonstrated that proteomics technology has advanced to the level where it can contribute to biomarker discovery, major drawbacks, such as large sample requirements (42–700 μg) and low proteome coverage (50–76 proteins), for small amounts of starting material (∼1 μg) persist. Because clinical samples are often available in limited quantities, in-depth analysis of minute amounts of material (<1 μg) necessitates advanced technologies with sufficient sensitivity and depth of coverage.Recently we demonstrated the applicability of nano-LC-FTICR MS in combination with the accurate mass and time (AMT) tag approach for proteomics characterization of ∼3,000 LCM-derived breast cancer cells (23). This study showed that proteome coverage was improved compared with conventional techniques. The AMT tag approach initially utilizes conventional LC-MS/MS measurements to establish a reference database of AMT tags specific for a particular proteome sample (e.g. breast cancer tissue). Each tag consists of a theoretical mass calculated from the peptide sequence, an LC normalized elution time (NET) value, and an indicator of quality. The AMT tag database serves as a “lookup table” for identifying peptides in subsequent quantitative LC-MS analyses. Substituting routine LC-MS/MS analyses (shotgun approach) with LC-FTICR MS analyses (AMT tag approach) significantly increases overall throughput and sensitivity while reducing sample requirements. Additionally quantitative intensity information related to the abundance of the protein can be discerned from these MS analyses (24). In the present study, we used the same strategy to analyze eight pools of tumor cells in duplicate or triplicate (resulting in 19 samples) derived from 51 fresh frozen primary invasive breast carcinomas that appeared to be either sensitive or resistant to tamoxifen treatment after recurrence. This work resulted in the identification of a putative protein profile associated with tamoxifen therapy resistance. In addition, the top discriminating protein of the putative profile, extracellular matrix metalloproteinase inducer (EMMPRIN), was validated in an independent patient cohort and was significantly associated with resistance to tamoxifen therapy and shorter time to progression upon tamoxifen treatment in recurrent breast cancer.