Smart Markers for Watershed-Based Cell Segmentation

Automated cell imaging systems facilitate fast and reliable analysis of biological events at the cellular level. In these systems, the first step is usually cell segmentation that greatly affects the success of the subsequent system steps. On the other hand, similar to other image segmentation problems, cell segmentation is an ill-posed problem that typically necessitates the use of domain-specific knowledge to obtain successful segmentations even by human subjects. The approaches that can incorporate this knowledge into their segmentation algorithms have potential to greatly improve segmentation results. In this work, we propose a new approach for the effective segmentation of live cells from phase contrast microscopy. This approach introduces a new set of “smart markers” for a marker-controlled watershed algorithm, for which the identification of its markers is critical. The proposed approach relies on using domain-specific knowledge, in the form of visual characteristics of the cells, to define the markers. We evaluate our approach on a total of 1,954 cells. The experimental results demonstrate that this approach, which uses the proposed definition of smart markers, is quite effective in identifying better markers compared to its counterparts. This will, in turn, be effective in improving the segmentation performance of a marker-controlled watershed algorithm.     

The apoptotic,cytotoxic and genotoxic effect of novel binuclear boron-fluoride complex on endometrial cancer

Endometrial cancer (EC) is one of the most common types of gynecologic cancer of the female genital tract; it considered being the fourth leading death factor among other types of cancer. Therefore, developing new anti-cancer agents are crucial for cancer treatment. Based on the potential of Schiff based complexes for the induction of apoptosis, Schiff base compounds, and their metal complexes displayed excellent anticancer properties. In this current study, antiproliferative activity of [L(BF₂)₂] as a novel binuclear boron-fluoride complex was examined to preliminary research in eight different cell lines, HELA, DU-145, PC3, DLD-1, ECC-1, PNT1-A, HT-29, and MCF-7, it was found to have a potent, suppressive effect on human endometrial adenocarcinoma cell line ECC-1. Based on this data, later investigated its apoptotic, cytotoxic, and genotoxic properties on human endometrial adenocarcinoma cell line ECC-1 in different concentrations. Apoptotic and cytotoxic tests such as single cell gel electrophoresis assay (comet assay), DNA fragmentation laddering, acridine orange test for DNA damage, and ELISA for apoptotic measurement was performed. We also gauged the oxidative status by evaluating total antioxidant status (TAS) and total oxidant status (TOS). Oxidative stress index (OSI) was calculated too. As a result [L(BF₂)₂] has been found to have a marvelous effect on ECC-1 cells, especially in damaging their DNA and cause a series of reactions lead to apoptosis. Taken together, it suggests that the [L(BF₂)₂] complex can induce the apoptotic pathway of endometrial cancer cells and is a possible candidate for future cancer treatment studies.     

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

Background and Aims

Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns.

Methods

Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden.

Key Results

The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden.

Conclusions

Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.Key words: Biogeography, Cypripedium calceolus, genome size, plastid microsatellites, population genetics, seed dispersal     

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain

Colidiarrhea and colienterotoxemia caused by F4⁺ and/or F18⁺ enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac⁺ non-ETEC vaccine candidate strain against challenge infection with F4ac⁺ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac⁺ non-ETEC strain (at day 0), and challenged with F4ac⁺ ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3⁺, CD45RA⁺, CD45RC⁺, CD21⁺, IgA⁺ and myeloid SWC3⁺ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3⁺, CD45RC⁺ and SWC3⁺ cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21⁺ cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac⁺ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.Key words: nonspecific/specific immunization, E. coli, gut immune cells, pigs.Porcine colidiarrhea and colienterotoxemia induced with F4⁺ and/or F18⁺ enterotoxigenic Escherichia coli (ETEC) strains are economically the most significant diseases of swine which account for moderate to high mortality rates and growth retardation, causing death of 5 million pigs per year in the World. Protection from ETEC is a constant challenge due to high genetic flexibility of this widespread bacterial organism.The virulence characteristics of ETEC are strongly dependent on the production of fimbrial adhesins and enterotoxins (Nagy and Fekete, 1999).The ability of adhesion of ETEC to intestinal wall is mainly due to the production of fimbriae. Enterotoxins produced by adherent ETEC strains act locally on enterocytes and stimulate increased water and electrolyte secretion and decreased fluid absorption. Several types of porcine ETEC are known today, including ETEC strain producing F18 fimbriae with their variants “ab” and “ac” (Bretschinger et al., 1990; Nagy and Fekete, 1999; Zang et al., 2007). The ETEC strains causing diarrhea mostly express F4 or F18 adhesins (Fairbrother et al., 2005; Zang et al., 2007). Nearly all known E. coli enterotoxin genes are produced by ETEC strains expressing either F4 or F18 fimbria. Zhang et al. (2007) have conclude that the dominant pathotypes causing diarrhea in weaned pigs are porcine ETEC strains expressing either F4 fimbria and heat-labile (LT) / heat stable (STb) toxins or LT/STb/EAST1 toxins, or F18 fimbria and STa/STb/Stx2e toxins. However, F18ab is more frequently associated with Shiga like toxin 2e, whereas F18ac is more frequently associated with enterotoxin STI (Cheng et al., 2005). Olasz et al. (2005) showed that the 200-kb plasmid, called pF18, contained the genes responsible for F18 fimbriae production.The curing of F18⁺ ETEC strain, i.e. loss of plasmid carrying the heat stable toxin genes in bacterial mutants has been performed by the plasmid transformation and conjugation following co-culturing of donor and recipient strains as reported earlier (Olasz et al., 2005). New vaccination strategies include the oral immunization of pigs with live avirulent nontoxigenic E. coli strains carrying the fimbrial adhesins F4 and/or F18 (Fairbrother et al., 2005).Receptors for F18ab and F18ac variants are increasingly produced up to the weaning age and the fimbriae F18ac seem to have more receptors around the ileal Peyer''s patches (Nagy et al., 1992). The colonization of the small intestine by an F18⁺ ETEC strain causes enterotoxemia. The typical clinical symptoms of the disease are neurological signs such as ataxia, convulsions and paralysis (Vögeli et al., 1996). It is well known that enterotoxic colibacillosis produces significant losses in two different age groups of pigs: first among newborn pigs and later at the postweaning age (Nagy and Fekete, 1999). The disease usually starts a few days after lacteal protection completely ceases (within the first 2 weeks after weaning), especially when weaning occurs at 3–4 weeks of age.Thus, the success of a vaccine against porcine colidiarrhea and colienterotoxemia depends upon applying it in the most efficient form at the optimal time and matching the right protective antigens with the type of virulence factors of ETEC present in the given animal population (Nagy and Fekete, 2005).The gut mucosal immune system contains specialized lymphoid tissues where environmental antigens are presented inducing B- and T-cell responses (Stokes et al., 1994). These responses are regulated by T cells and cytokines and they lead to plasma cell differentiation and the secretion of IgA antibodies onto intestinal mucosal surfaces.The aggregated lymphoid tissue such as Peyer’s patches and solitary lymphoid cells in the lamina propria both play important roles in the induction and regulation of immune responses in the gut associated lymphoid tissues (GALT) (Lacković et al.,1997b). Such organization of the GALT may provide immune protection at mucosal surfaces where the infection actually occurs (McGhee et al., 1992). Bertschinger et al. (2000) demonstrated the protective effects of a live oral vaccine containing F18 fimbria against porcine postweaning diarrhea and oedema disease.In this study we have examined the distribution and quantitative patterns of the subsets of T and B cells as well as of macrophages and secretory IgA⁺ plasma cells within GALT compartments of 4 weeks old pigs perorally immunized with an attenuated F18ac⁺ non-ETEC vaccine candidate strain against porcine colienterotoxemia. Additionally, we have evaluated adjuvanticity of levamisole in the combination with the experimental vaccine and its immunostimulatory effect when applied as an immune response modifier (IRM). Levamisole (2,3,5,6-tetrahydro-6-phenylimidazole thiazole), was originally described as a highly effective anti-helminthic compound (Thienpont et al., 1966). Subsequent studies have established its ability to restore and enhance depressed immune responses in domestic food animals and to act as an effective adjuvant for parenteral and oral vaccines (Mulcahy and Quinn, 1986; Jenkins and Hurdle, 1989; Božić et al., 2002).