An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease.MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC₅₀ and K_i values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot.ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC₅₀ (29 nM) and K_i (15 nM) values. TF inhibited only seven out of 61 human kinases tested (> 70% inhibition). Incubation of LNCaP cells with 50 μM TF for 48 h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis.General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.     

Glucosinolate contents in maca (Lepidium peruvianum Chacón) seeds, sprouts, mature plants and several derived commercial products

Several products derived from processed maca hypocotyls (Lepidium peruvianum Chacón, previously known asL. meyenii Walp.) were surveyed for glucosinolate content and quantified by HPLC analysis. These included pills, capsules, flour, liquor, tonic and mayonnaise. Different plant organs such as fresh hypocotyls and leaves, seeds, dry hypocotyls, and sprouts were also included in the survey. The most abundant glucosinolates detected in fresh and dry hypocotyls and leaves were the aromatic glucosinolates, benzylglucosinolate (glucotropaeolin) and p-methoxybenzylglucosinolate. Maca seeds and sprouts differed in profile from hypocotyls and leaves due to the modification of benzylglucosinolate. No glucosinolates were detected in liquor and tonic, while mayonnaise had only trace amounts of those glucosinolates. It had instead allylglucosinolate (sinigrin), which is an aliphatic glucosinolate. The pills, capsules and flour had the same glucosinolates as those observed in hypocotyls, but in variable amounts. The richest sources of glucosinolates were seeds, fresh hypocotyls and sprouts, in that order.     

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation ( Ds ), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep⁺) and non-replicating (rep⁻) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep⁺) or mutant, inactive derivatives of these genes (rep⁻). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds -vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 × 10⁵-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.     

Diversity of North American map and sawback turtles (Testudines: Emydidae: Graptemys)

Map turtles of the genus Graptemys are native to North America, where a high degree of drainage endemism is believed to have shaped current diversity. With 14 species and one additional subspecies, Graptemys represents the most diverse genus in the family Emydidae. While some Graptemys species are characterized by pronounced morphological differences, previous phylogenetic analyses have failed yet to confirm significant levels of genetic divergence for many taxa. As a consequence, it has been debated whether Graptemys is taxonomically inflated or whether the low genetic divergence observed reflects recent radiations or ancient hybridization. In this study, we analysed three mtDNA blocks (3228 bp) as well as 12 nuclear loci (7844 bp) of 89 specimens covering all species and subspecies of Graptemys. Our analyses of the concatenated mtDNA sequences reveal that the widespread G. geographica constitutes the sister taxon of all other Graptemys species. These correspond to two clades, one comprised of all broad‐headed Graptemys species and another clade containing the narrow‐headed species. Most species of the broad‐headed clade are reciprocally monophyletic, except for G. gibbonsi and G. pearlensis, which are not differentiated. By contrast, in the narrow‐headed clade, many currently recognized species are not monophyletic and divergence is significantly less pronounced. Haplotype networks of phased nuclear loci show low genetic divergence among taxa and many shared haplotypes. Principal component analyses using coded phased nuclear DNA sequences revealed eight distinct clusters within Graptemys that partially conflict with the terminal mtDNA clades. This might be explained by male‐mediated gene flow across drainage basins and female philopatry within drainage basins. Our results support that Graptemys is taxonomically oversplit and needs to be revised.     

Non-condensation of one segment of a chromosome No. 2 in a male with an otherwise normal karyotype (and severe hypospadias)

Summary A child with severe hypospadias is presented, whose karyotype showed in about 11% of mitoses of peripheral blood one member of chromosome pair No. 2 with a non-condensed region near the centromere. The non-condensed segment does not show late replication, however, it is situated very close to the late replicating segment of the long arms of chromosome No. 2. The nature and possible implications of this kind of aberration are discussed. It is held that non-condensation can produce localized chromosome breaks by a mechanism possibly different from any of the classical breakage mechanisms.     

Juxtaposition of System Dynamics and Agent-Based Simulation for a Case Study in Immunosenescence

Advances in healthcare and in the quality of life significantly increase human life expectancy. With the aging of populations, new un-faced challenges are brought to science. The human body is naturally selected to be well-functioning until the age of reproduction to keep the species alive. However, as the lifespan extends, unseen problems due to the body deterioration emerge. There are several age-related diseases with no appropriate treatment; therefore, the complex aging phenomena needs further understanding. It is known that immunosenescence is highly correlated to the negative effects of aging. In this work we advocate the use of simulation as a tool to assist the understanding of immune aging phenomena. In particular, we are comparing system dynamics modelling and simulation (SDMS) and agent-based modelling and simulation (ABMS) for the case of age-related depletion of naive T cells in the organism. We address the following research questions: Which simulation approach is more suitable for this problem? Can these approaches be employed interchangeably? Is there any benefit of using one approach compared to the other? Results show that both simulation outcomes closely fit the observed data and existing mathematical model; and the likely contribution of each of the naive T cell repertoire maintenance method can therefore be estimated. The differences observed in the outcomes of both approaches are due to the probabilistic character of ABMS contrasted to SDMS. However, they do not interfere in the overall expected dynamics of the populations. In this case, therefore, they can be employed interchangeably, with SDMS being simpler to implement and taking less computational resources.