Proteome analyses of hepatocellular carcinoma

Proteomics has evolved into a powerful and widely used bioanalytical technique in the study of cancer, especially hepatocellular carcinoma (HCC). In this review, we provide an up to date overview of feasible proteome-analytical techniques for clinical questions. In addition, we present a broad summary of proteomic studies of HCC utilizing various technical approaches for the analysis of samples derived from diverse sources like HCC cell lines, animal models, human tissue and body fluids

Reproductive biology of dry grassland specialist Ranunculus illyricus L. and Its implications for conservation

SIMPLE SUMMARY: The Ranunculus illyricus—Illyrian buttercup—is threatened with extinction in many countries and measures should be taken to protect it. In order to increase the effectiveness of such measures, it is necessary to know the methods of propagation and to evaluate their efficiency. R. illyricus reproduces generatively by seed and vegetatively by clusters of progeny tubers. The method and potential of vegetative propagation are described here for the first time and compared with the potential and actual effectiveness of generative propagation. Both the generative and vegetative propagation methods should be used to strengthen existing populations and create replacements. ABSTRACT: Ranunculus illyricus, a component of xerothermic grasslands, is a declining species and deserves active conservation treatments in many countries preceded by studies on the biology of its reproduction. So far, our knowledge of R. illyricus, a species with two modes of reproduction, has been fragmentary. The purpose of the studies presented here was to describe the annual development cycle of R. illyricus with particular emphasis on the production of underground tuber clusters that serve as vegetative propagation. Based on three-year-long observations in an ex situ collection, the efficiency of vegetative propagation was estimated and compared with the efficiency of generative propagation. It was found that in 3 years the best clones could produce up to 57 progeny clusters followed by flowering specimens in the first season. Meanwhile, the high potential for generative reproduction was suppressed by many limitations including fruit setting, the germination capacity of seeds, seedling survival rate, and additionally, the first flowering plant was observed only in the third year. It seems that the efficiency of vegetative propagation of this species can be higher than the efficiency of generative propagation. Moreover, vegets bloomed in the first year after emergence, whereas the first plant of generative origin was observed to bloom only after 3 years. A large proportion of individuals of vegetative origin can negatively affect the genetic diversity of the population but their survival rate against competing plants is higher. To enhance the existing populations or to create new ones, it would be best to use plants derived from clonal propagation of genets carried out in ex situ conditions

Conservation of Primula farinosa in Poland with respect to the genetic structure of populations

The bird’s-eye primrose (Primula farinosa L.) is an endangered species in Poland. The sole remaining, and critically endangered, population of approximately 300 flowering plants is located in the Beskid Sądecki Mountains (Jaworki, Poland). The genetic investigation was performed using highly variable microsatellite markers on a total of four populations: the Polish population and its three nearest known Slovak counterparts. We hypothesize that the Polish population is a relic of the previously much wider Central European mountain/lowland range and is thus genetically distinct from the rest of the Slovak stock. Clarification of this issue is needed before active protection interventions such as artificial supplementation can be carried out. Our results, particularly those from STRUCTURE Bayesian clustering, showed clear population structure within the dataset: all three Slovak populations were dominated by one genetic group, while the Polish population comprised mostly individuals dominated by the second genetic group. Only limited gene flow was observed between the Polish and Slovak populations. This demonstrates the distinct genetic makeup of the Jaworki population, which is probably the result of prolonged isolation from the rest of the P. farinosa range and admixture of genes from various lineages. Their origin could be determined by including plant material from the rest of the P. farinosa range, i.e., Scandinavian, Baltic, and Alpine/Carpathian locations, in a future study. The immediate practical application of our results is the recommendation that all supplementation interventions to the Jaworki population must be limited to plants produced from locally collected seeds

Systematic analysis of synergistic proteome modulations in a drug combination of cisplatin and MLN4924

Chemotherapeutic treatment regimens often take advantage of synergistic effects of drug combinations. Anticipating that synergistic effects on the cell biological level likely manifest on the proteome level, the analysis of proteome modulations represents an appropriate strategy to study drug combinations on a molecular level. More specifically, the detection of single proteins exhibiting synergistic abundance changes could be helpful to shed light on key molecules, which contribute in mechanisms facilitating the synergistic interaction and therefore represent potential targets for specific therapeutic approaches. In the reported study we aimed to provide evidence for this assumption and investigated the drug combination of cisplatin and the neddylation inhibitor MLN4924 in HCT-116 cells via cell biological analyses and mass spectrometry-based quantitative proteomics. From 1789 proteins quantified with two unique peptides, activated RNA polymerase II transcriptional coactivator p15 (SUB1) was highlighted as the most synergistically regulated protein using a synergistic scoring approach. Western blotting and analyses of cellular processes associated with this protein (DNA damage, oxidative stress and apoptosis) revealed supporting evidence for the synergistic regulation. Whereas the distinct role of SUB1 in the investigated drug combination needs to be elucidated in future studies, the presented results demonstrated the benefit and feasibility of synergistic scoring of proteome alterations to highlight proteins that likely contribute to the underlying molecular mechanisms of synergistic effects. Data are available via ProteomeXchange with identifier PXD009185

AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes

Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11–19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression

Can ceylon leadwort (Plumbago zeylanica L.) acclimate to lead toxicity? - studies of photosynthetic apparatus efficiency

Ceylon leadwort (Plumbago zeylanica) is ornamental plant known for its pharmacological properties arising from the abundant production of various secondary metabolites. It often grows in lead polluted areas. The aim of presented study was to evaluate the survival strategy of P. zeylanica to lead toxicity via photosynthetic apparatus acclimatization. Shoots of P. zeylanica were cultivated on media with different Pb concentrations (0.0, 0.05, and 0.1 g Pb∙l−1). After a four-week culture, the efficiency of the photosynthetic apparatus of plants was evaluated by Chl a fluorescence measurement, photosynthetic pigment, and Lhcb1, PsbA, PsbO, and RuBisCo protein accumulation, antioxidant enzymes activity, and chloroplast ultrastructure observation. Plants from lower Pb concentration revealed no changes in photosynthetic pigments content and light-harvesting complex (LHCII) size, as well as no limitation on the donor side of Photosystem II Reaction Centre (PSII RC). However, the activity and content of antioxidant enzymes indicated a high risk of limitation on the acceptor side of Photosystem I. In turn, plants from 0.1 g Pb∙l−1 showed a significant decrease in pigments content, LHCII size, the amount of active PSII RC, oxygen-evolving complex activity, and significant remodeling of chloroplast ultrastructure indicated limitation of PSII RC donor side. Obtained results indicate that P. zeylanica plants acclimate to lead toxicity by Pb accumulation in roots and, depending on Pb concentration, by adjusting their photosynthetic apparatus via the activation of alternative (cyclic and pseudocyclic) electron transport pathways

Individual Profiling of Circulating Tumor Cell Composition and Therapeutic Outcome in Patients with Hepatocellular Carcinoma

AbstractBACKGROUND AND AIMS: Circulating tumor cells (CTCs) have been proposed as a monitoring tool in patients with solid tumors. So far, automated approaches are challenged by the cellular heterogeneity of CTC, especially the epithelial-mesenchymal transition. Recently, Yu and colleagues showed that shifts in these cell populations correlated with response and progression, respectively, to chemotherapy in patients with breast cancer. In this study, we assessed which non-hematopoietic cell types were identifiable in the peripheral blood of hepatocellular carcinoma (HCC) patients and whether their distribution during treatment courses is associated with clinical characteristics. METHODS: Subsequent to few enrichment steps, cell suspensions were spun onto glass slides and further characterized using multi-immunofluorescence staining. All non-hematopoietic cells were counted and individual cell profiles were analyzed per patient and treatment. RESULTS:We detected a remarkable variation of cells with epithelial, mesenchymal, liver-specific, and mixed characteristics and different size ranges. The distribution of these subgroups varied significantly between different patient groups and was associated with therapeutic outcome. Kaplan-Meier logrank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time to progression (1 vs 15 months; P = .03; hazard ratio = 0.18; 95%confidence interval = 0.01-2.75). CONCLUSIONS: Our data suggest that different CTC populations are identifiable in peripheral blood of HCC patients and, for the first time in HCC, that these individual cell type profiles may have distinct clinical implications. The further characterization and analysis of patients in this ongoing study seems to be warranted

Comparison of pulmonary and systemic NO- and PGI_{2}-dependent endothelial function in diabetic mice

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (Kf,c) and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO2− were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO2− generation, and production of 6-keto-PGF1α were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression