Dissecting Tn5 transposition using HIV-1 integrase diketoacid inhibitors

Diketoacid (DKA) compounds have been shown to inhibit HIV-1 integrase by a mechanism that involves sequestration of the active site metals. Because HIV-1 integrase and Tn5 transposase have similar active site architectures and catalytic mechanisms, we investigated whether DKA analogues would inhibit Tn5 transposase activity and provide a model system to explore the mechanisms of action of these inhibitors. A screen of several hundred DKA analogues identified several with activity against Tn5 Tnp. Six DKA inhibitors used in this study manifested a variety of effects on different transposition steps suggesting that different analogues may have different binding contacts with transposase. All DKA compounds inhibited paired end complex (PEC) formation in which the nucleoprotein complex required for catalysis is assembled. Dissociation of PECs by some DKA compounds indicates that these inhibitors can decrease PEC stability. Four DKA compounds inhibited the two cleavage steps releasing transposon DNA from flanking DNA, and one of these four compounds preferentially inhibited the second cleavage step. The differential effect of this inhibitor on the second cleavage event indicates that cleavage of the two transposon-donor DNA boundaries is a sequential process requiring a conformational change. The requirement for a conformational change between cleavage events was also demonstrated by the inability of transposase to perform second cleavage at 25 degrees C. Finally, all six compounds inhibit strand transfer, the final step of Tn5 transposition. Two of the compounds that inhibited strand transfer have no effect on DNA cleavage. The strand transfer inhibition properties of various DKA compounds was sensitive to the structure of the 5'-non-transferred strand, suggesting that these compounds bind in or near the transposase active site. Other results that probe compound binding sites include the effects of active site mutations and donor DNA on DKA compound inhibition activities. Thus, DKA inhibitors will provide an important set of tools to investigate the mechanism of action of transposases and integrases.     

The Role of Energy in the Emergence of Biology from Chemistry

Any scenario of the transition from chemistry to biology should include an ??energy module?? because life can exist only when supported by energy flow(s). We addressed the problem of primordial energetics by combining physico-chemical considerations with phylogenomic analysis. We propose that the first replicators could use abiotically formed, exceptionally photostable activated cyclic nucleotides both as building blocks and as the main energy source. Nucleoside triphosphates could replace cyclic nucleotides as the principal energy-rich compounds at the stage of the first cells, presumably because the metal chelates of nucleoside triphosphates penetrated membranes much better than the respective metal complexes of nucleoside monophosphates. The ability to exploit natural energy flows for biogenic production of energy-rich molecules could evolve only gradually, after the emergence of sophisticated enzymes and ion-tight membranes. We argue that, in the course of evolution, sodium-dependent membrane energetics preceded the proton-based energetics which evolved independently in bacteria and archaea.     

Population structure of the Monocelis lineata (Proseriata, Monocelididae) species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene

Monocelis lineata consists of a complex of sibling species, widespread in the Mediterranean and Atlantic Ocean. Previous genetic analysis placed in evidence at least four sibling species. Nevertheless, this research was not conclusive enough to fully resolve the complex or to infer the phylogeny/phylogeography of the group. We designed specific primers aiming at obtaining partial sequences of the mtDNA gene Cytochrome c Oxidase subunit I (COI) of M. lineata, and have identified 25 different haplotypes in 32 analyzed individuals. The dendrogram generated by Neighbor-Joining analysis confirmed the differentiation between Atlantic and Mediterranean siblings, as well as the occurrence of at least two Mediterranean sibling species. Thus validated, the method here presented appears as a valuable tool in population genetics and biodiversity surveys on the Monocelis lineata complex.     

Seasonal abundance and feeding patterns of copepods Temora longicornis, Centropages hamatus and Acartia spp. in the White Sea (66��N)

We have studied the seasonal dynamics of abundance and feeding characteristics of three species of calanoid copepods (Acartia spp., Centropages hamatus and Temora longicornis) in the White Sea from the surface water layer (0–10 m), in order to assess their role in the pelagic food web and to determine the major factors governing their population dynamics during the productive season. These species dominated in the upper water layer (0–10 m) from June through September, producing up to 3 generations per year. Data on the food spectra revealed all species to be omnivorous; but some inter- and intraspecific differences were observed. Generally, copepods consumed diatoms, dinoflagellates and microzooplankton. The omnivory index ‘UC’ (i.e., fatty acid unsaturation coefficient) varied from 0.2 to 0.6, which implied ingestion of phytoplankton. The different degree of selectivity on the same food items by the studied species was observed, and therefore, successful surviving strategy with minimal overlapping could be assumed. In total, the populations of the three studied copepod species grazed up to 2.15 g C m⁻² day⁻¹ and released up to 0.68 g C m⁻² day⁻¹ in faecal pellets. They consumed up to 50% of particulate organic carbon, or up to 85% of phytoplankton standing stock (in terms of Chl. a), and thus played a significant role in the transformation of particulate organic matter. Seasonal changes in abundance of the studied species depended mostly on water temperature in the early summer, but were also affected by food availability (Chl. a concentration) during the productive season.     

Same modulation but different starting points: performance modulates age differences in inferior frontal cortex activity during word-retrieval

The neural basis of word-retrieval deficits in normal aging has rarely been assessed and the few previous functional imaging studies found enhanced activity in right prefrontal areas in healthy older compared to younger adults. However, more pronounced right prefrontal recruitment has primarily been observed during challenging task conditions. Moreover, increased task difficulty may result in enhanced activity in the ventral inferior frontal gyrus (vIFG) bilaterally in younger participants as well. Thus, the question arises whether increased activity in older participants represents an age-related phenomenon or reflects task difficulty effects. In the present study, we manipulated task difficulty during overt semantic and phonemic word-generation and used functional magnetic resonance imaging to assess activity patterns in the vIFG in healthy younger and older adults (N = 16/group; mean age: 24 vs. 69 years). Both groups produced fewer correct responses during the more difficult task conditions. Overall, older participants produced fewer correct responses and showed more pronounced task-related activity in the right vIFG. However, increased activity during the more difficult conditions was found in both groups. Absolute degree of activity was correlated with performance across groups, tasks and difficulty levels. Activity modulation (difficult vs. easy conditions) was correlated with the respective drop in performance across groups and tasks. In conclusion, vIFG activity levels and modulation of activity were mediated by performance accuracy in a similar way in both groups. Group differences in the right vIFG activity were explained by performance accuracy which needs to be considered in future functional imaging studies of healthy and pathological aging.     

Cellular architecture and migration behavior of fibroblast cells on polyhydroxyoctanoate (PHO): A natural polymer of bacterial origin

Biodegradable and biocompatible novel materials of natural origin are gaining more and more attention in recent years. These so called biopolymers, characterized by their biointegrity and biocompatibility, find completely new and promising applications in biomedical sciences. The presented work focuses on the medium chain length elastomeric polyhydroxyalkanoate biopolymer—polyhydroxyoctanoate (PHO). This biopolymer is fully biodegradable without formation of harmful byproducts.We investigated PHO's physical properties with nanoindentation technique and scratch testing to determine Young's modulus and friction coefficient. Further, the work focused on the impact of PHO, used as growth substrate, on the physiology and morphology of mouse embryonic fibroblast cells (MEF 3T3). Application of fluorescent staining protocols and advanced microscopic techniques allowed to study the morphological changes in the cytoskeletons of cells grown on PHO and also gave an insight into their migration strategies on the polymer surface. We found that PHO exhibits no cellular cytotoxicity, similarly to a glass substrate. MEF cells spread better on glass surface than on each tested PHO substrate though there was almost no difference between PHO substrates cast from different solvents. However, a detailed analysis of actin and microtubule cytoskeletal architecture reveals changes in the density of actin and microtubular networks. Migration of MEF cells on PHO substrates was slower than on the glass substrate. To elucidate the molecular mechanisms of observed changes in cytoskeletal architecture and migration parameters can be of special interest for future medical application of PHO polymer.     

The secretion antigen SA5K has a role in the adaptation of Mycobacterium bovis bacillus Calmette-Guérin to intracellular stress and hypoxia

The Mycobacterium tuberculosis TB8.4 (Rv1174c) gene encodes a secreted protein of 8.4 kDa (TB8.4) which has been suggested to be involved in reactivation of dormant mycobacteria. We have previously reported that inactivation of an identical gene (sa5k) in Mycobacterium bovis BCG causes impaired ability of the mutant strain (BCGsa5k::aph) to grow inside human macrophages. This study aimed to investigate the role of TB8.4 in the reactivation of aged cultures of BCG as well as the role of the sa5k gene in the resistance of BCG to intracellular stress conditions and adaptation to hypoxia. Although when added to aged cultures of BCG, TB8.4 caused a statistically significant increase in the number of colony-forming units, a similar effect was obtained in cultures incubated with BSA, suggesting a non-specific growth stimulation by TB8.4. Compared to parental BCG, the BCGsa5k::aph strain showed an increased susceptibility to reactive oxygen and nitrogen intermediates and to acid stress and an impaired ability to adapt to reduced O2 concentrations, when tested in the oxygen-limited Wayne culture system. These results suggest that the product of the sa5k gene (SA5K protein) has a role in both resistance of BCG to intracellular stress and in its adaptation to hypoxia.     

131I-MIBG therapy in the treatment of pheochromocytoma in children--own experiences

Three cases of pheochromocytoma in children/adolescents or young adults treated by 131I-MIBG are presented in this study. In one patient 131I-MIBG was administrated after ineffective surgical treatment and chemotherapy of a benign retroperitoneal tumor, whereas in two other patients 131I-MIBG therapy was carried out because of malignant pheochromocytoma dissemination. In a child with retroperitoneal paraganglioma decrease of tumor size and its fibrosis after 131I-MIBG therapy allowed radical surgery and complete recovery. In two other cases partial remission was achieved. All patients showed a good subjective response with improvement of the general condition and better blood pressure control. In two children adverse reactions such as leucopenia, hypothyroidism or hypogonadism were observed. The presented data confirm effectiveness and acceptable tolerance of 131I-MIBG treatment in pheochromocytoma, what is very important in pediatric patients.