Synthesis and Microdiffraction at Extreme Pressures and Temperatures

High pressure compounds and polymorphs are investigated for a broad range of purposes such as determine structures and processes of deep planetary interiors, design materials with novel properties, understand the mechanical behavior of materials exposed to very high stresses as in explosions or impacts. Synthesis and structural analysis of materials at extreme conditions of pressure and temperature entails remarkable technical challenges. In the laser heated diamond anvil cell (LH-DAC), very high pressure is generated between the tips of two opposing diamond anvils forced against each other; focused infrared laser beams, shined through the diamonds, allow to reach very high temperatures on samples absorbing the laser radiation. When the LH-DAC is installed in a synchrotron beamline that provides extremely brilliant x-ray radiation, the structure of materials under extreme conditions can be probed in situ. LH-DAC samples, although very small, can show highly variable grain size, phase and chemical composition. In order to obtain the high resolution structural analysis and the most comprehensive characterization of a sample, we collect diffraction data in 2D grids and combine powder, single crystal and multigrain diffraction techniques. Representative results obtained in the synthesis of a new iron oxide, Fe₄O₅ ¹ will be shown.     

Impact of functionalized coligands on the pharmacokinetics of 99mTcIII '4+1' mixed-ligand complexes conjugated to bombesin

Bombesins (BN) containing ^99mTc ‘4 + 1’ complexes may be useful to detect tumors expressing the gastrin-releasing peptide receptor (GRPR). Derivatives of the formula [^99mTc(NS₃R)(L2-BN_st)] were synthesized, in which Tc(III) is coordinated by an isocyanide L2-BN_st bearing the peptide (BN_st = βAla-βAla-Gln-Trp-Ala-Val-Gly-His-Cha-Nle-NH₂) and a tetradentate chelator NS₃R. NS₃R consists of 2,2′,2″-nitrilotriethanethiol (NS₃) bearing a crown ether (NS₃crown), an aliphatic amine (NS₃en) and a tricarboxylic acid (NS₃(COOH)₃). Non-radioactive Re compounds were prepared and analysed by electrospray ionization mass spectrometry. The structural similarity to the ^99mTc conjugates was demonstrated by their identical HPLC elution profiles. The lipophilicity of [^99mTc(NS₃R)(L2-BN_st)] decreased depending on the coligands NS₃crown (log D_O/W, pH = 7.4, 0.98 ± 0.11), NS₃en (− 0.49 ± 0.07) and NS₃(COOH)₃ (− 2.01 ± 0.09). Biodistribution in normal rats was characterized by an increasing kidney uptake and a decreasing uptake into the liver corresponding to the reduced lipophilicity of the conjugates. The pancreatic uptake expressed by the organ/blood ratio of standardized uptake values at 60 min p.i. in rats was 8.6 ± 1.2 for [^99mTc(NS₃en)(L2-BN_st)] and higher compared to the other conjugates. The pancreas/liver ratio of the SUV at 60 min p.i. in rats was highest for [^99mTc(NS₃(COOH)₃)(L2-BN_st)] at 8.4 ± 1.3. [^99mTc(NS₃en)(L2-BN_st)] was further studied in tumor-bearing mice and its pancreas/blood and pancreas/liver ratios were lower, however the pancreas/kidney ratios were higher in mice compared to rats. The activity uptake of [^99mTc(NS₃en)(L2-BN_st)] into the PC-3 tumor xenografts was low (%ID/g: 0.83 ± 0.18 at 60 min; SUV: 0.21 ± 0.05 at 60 min) but specific.     

Entropy versus aromaticity in the conformational dynamics of aromatic rings

Comparison of the results of Car-Parrinello molecular dynamics simulations of isolated benzene, pyrimidine and 1,2,4-triazine molecules reveals that the unusually low population of planar geometry of the benzene ring is caused by entropy effects despite its high aromaticity. The decrease in symmetry of the molecule results in smaller changes in entropy and Gibbs free energy due to out-of-plane deformations of the ring, leading to an increase in the population of planar geometry of the ring. This leads to differences in the topology of potential energy and Gibbs free energy surfaces.

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.     

Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil

Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial community in soil. The current study provides information on the response of soil bacterial, archaeal and fungal communities during the degradation of three priority pollutants and contributes to a knowledge base that can inform the development of effective bioremediation strategies for contaminated sites.     

A Comparative and Phylogenetic Study of the Ditylenchus dipsaci,Ditylenchus destructor and Ditylenchus gigas Populations Occurring in Poland

The genus Ditylenchus contains more than 80 recognized nematode species with a very wide host range. The most serious species are Ditylenchus dipsaci and Ditylenchus destructor. Populations of D. dipsaci species complex were collected from Allium cepa, Cichorium endivia and Phlox paniculata in Poland. The Ditylenchus gigas population was collected from Vicia faba minor, and populations of D. destructor, from Solanum tuberosum spp. tuberosum. Analyses of the rDNA sequences spanning both ITS1 and ITS2 fragment regions were carried out on the collected populations. The obtained DNA sequences were compared with those DNA sequences deposited in GenBank of populations isolated in other countries. Phylogenetic analysis was performed using the data obtained from the DNA sequence comparisons. The results indicated that there is no clear distinction between European and non‐European populations within D. dipsaci. The results also showed no clear distinction between populations isolated from different host plant species, including populations found in Poland. The populations of D. destructor described here constitute a common group together with American and Chinese populations belonging to the haplotype C of the D. destructor species. On the other hand, the D. gigas population was localized separately from those populations that have been described up until now, from Europe and Africa. This is also the first report on the occurrence of D. gigas in Poland.     

Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.