Postmortem whole-body CT angiography: evaluation of two contrast media solutions

OBJECTIVE: The objective of our study was to establish a standardized procedure for postmortem whole-body CT-based angiography with lipophilic and hydrophilic contrast media solutions and to compare the results of these two methods. MATERIALS AND METHODS: Minimally invasive postmortem CT angiography was performed on 10 human cadavers via access to the femoral blood vessels. Separate perfusion of the arterial and venous systems was established with a modified heart-lung machine using a mixture of an oily contrast medium and paraffin (five cases) and a mixture of a water-soluble contrast medium with polyethylene glycol (PEG) 200 in the other five cases. Imaging was executed with an MDCT scanner. RESULTS: The minimally invasive femoral approach to the vascular system provided a good depiction of lesions of the complete vascular system down to the level of the small supplying vessels. Because of the enhancement of well-vascularized tissues, angiography with the PEG-mixed contrast medium allowed the detection of tissue lesions and the depiction of vascular abnormalities such as pulmonary embolisms or ruptures of the vessel wall. CONCLUSION: The angiographic method with a water-soluble contrast medium and PEG as a contrast-agent dissolver showed a clearly superior quality due to the lack of extravasation through the gastrointestinal vascular bed and the enhancement of soft tissues (cerebral cortex, myocardium, and parenchymal abdominal organs). The diagnostic possibilities of these findings in cases of antemortem ischemia of these tissues are not yet fully understood.

Variation of the mechanical properties of PMMA to suit osteoporotic cancellous bone

Poly(methyl methacrylate) (PMMA) is by far the most frequently used bone substitute material for vertebroplasty. However, there are serious complications, such as cement leakage and an increased fracture rate of the adjacent vertebral bodies. The latter may be related to the mechanical properties of the augmented segment within the osteoporotic spine. A possible counter-measure is prophylactic augmentation at additional levels, but this aggravates the risk for the patient. Introduction of pores is a possible method to reduce the inherent high stiffness of PMMA. This study investigates the effect of porosity on the mechanical properties of PMMA bone cement. Different fractions of a highly viscous liquid were mixed into the PMMA during preparation. An open-porous material with adjustable mechanical properties resulted after removal of the aqueous phase. Different radiopacifiers were admixed to investigate their suitability for vertebroplasty. The final material was characterized mechanically by compressive testing, microscopically and radiologically. In addition, the monomer release subsequent to hardening was measured by means of gas chromatography. The Young's modulus in compression could be varied between 2800 +/- 70 MPa and 120 +/- 150 MPa, and the compression ultimate strength between 170 +/- 5 MPa and 8 +/- 9 MPa for aqueous fractions ranging between 0 and 50% of volume. Only a slight decrease of the Young's modulus and small changes of ultimate strength were found when the mixing time was increased. An organic hydrophilic and lipophilic radiopacifier led to a higher Young's modulus of the porous material; however, the ultimate strength was not significantly affected by adding different radiopacifiers to the porous cement. The radiopacity was lost after washing the aqueous phase out of the pores. No separation occurred between the aqueous and the PMMA phase during injection into an open porous ceramic material. The monomer released was found to increase for increasing aqueous fractions, but remained comparable in magnitude to standard PMMA. This study demonstrates that a conventional PMMA can be modified to obtain a range of mechanical properties, including those of osteoporotic bone.

UPLC-ESI-TOFMS-based metabolomics and gene expression dynamics inspector self-organizing metabolomic maps as tools for understanding the cellular response to ionizing radiation

Global transcriptomic and proteomic profiling platforms have yielded important insights into the complex response to ionizing radiation (IR). Nonetheless, little is known about the ways in which small cellular metabolite concentrations change in response to IR. Here, a metabolomics approach using ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry was used to profile, over time, the hydrophilic metabolome of TK6 cells exposed to IR doses ranging from 0.5 to 8.0 Gy. Multivariate data analysis of the positive ions revealed dose- and time-dependent clustering of the irradiated cells and identified certain constituents of the water-soluble metabolome as being significantly depleted as early as 1 h after IR. Tandem mass spectrometry was used to confirm metabolite identity. Many of the depleted metabolites are associated with oxidative stress and DNA repair pathways. Included are reduced glutathione, adenosine monophosphate, nicotinamide adenine dinucleotide, and spermine. Similar measurements were performed with a transformed fibroblast cell line, BJ, and it was found that a subset of the identified TK6 metabolites were effective in IR dose discrimination. The GEDI (Gene Expression Dynamics Inspector) algorithm, which is based on self-organizing maps, was used to visualize dynamic global changes in the TK6 metabolome that resulted from IR. It revealed dose-dependent clustering of ions sharing the same trends in concentration change across radiation doses. "Radiation metabolomics," the application of metabolomic analysis to the field of radiobiology, promises to increase our understanding of cellular responses to stressors such as radiation.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Two-dimensional supramolecular polymers

One of the biggest issues of modern materials science is developing of strategies to create large and ordered assemblies in the form of discrete nanoscale objects. Oligopyrenotides (OPs) represent novel class of amphiphilic molecules which tend to self-assemble forming highly ordered structures. As has been already shown OPs are able to form 1D («rod-like») supramolecular polymer [1]. Since programmed arraying of polyaromatic hydrocarbons in structurally defined objects could offer enhanced performance over the individual components, prediction and controlling of their spatial arrangement remains challenging. Herein we demonstrate that certain changes to design of pyrene’s molecular core allow Py3 form 2D supramolecular assemblies («nanosheets») instead of 1D. Two dimensional supramolecular polymers are attractive objects due to their exceptional properties which originate from in-plan alignment of molecular units in the sheets with constant thickness ~ 2 nm [2]. These assemblies have high degree of internal order: the interior consists of hydrophobic pyrenes and alkyl chains, whereas the exterior exists as a net of hydrophilic, negatively charged phosphates. The Py3 units are hold up by non-covalent interactions what makes these assemblies totally reversible. Moreover the polymerization occurs via nucleation-elongation mechanism. To study Py3 self-assembly, we carried out whole set of spectroscopic (UV/vis, fluorescence, DLS) and microscopic experiments (AFM)

Two-dimensional supramolecular polymers

One of the biggest issues of modern materials science is developing of strategies to create large and ordered assemblies in the form of discrete nanoscale objects. Oligopyrenotides (OPs) represent novel class of amphiphilic molecules which tend to self-assemble forming highly ordered structures. As has been already shown OPs are able to form 1D («rod-like») supramolecular polymer [1]. Since programmed arraying of polyaromatic hydrocarbons in structurally defined objects could offer enhanced performance over the individual components, prediction and controlling of their spatial arrangement remains challenging. Herein we demonstrate that certain changes to design of pyrene’s molecular core allow Py3 form 2D supramolecular assemblies («nanosheets») instead of 1D. Two dimensional supramolecular polymers are attractive objects due to their exceptional properties which originate from in-plan alignment of molecular units in the sheets with constant thickness ~ 2 nm [2]. These assemblies have high degree of internal order: the interior consists of hydrophobic pyrenes and alkyl chains, whereas the exterior exists as a net of hydrophilic, negatively charged phosphates. The Py3 units are hold up by non-covalent interactions what makes these assemblies totally reversible. Moreover the polymerization occurs via nucleation-elongation mechanism. To study Py3 self-assembly, we carried out whole set of spectroscopic (UV/vis, fluorescence, DLS) and microscopic experiments (AFM)

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Bimanual anterior segment revision surgery for anterior capsule contraction syndrome associated with anterior flexion of intraocular lens haptics

PURPOSE To report the incidence of anterior capsule contraction syndrome (ACCS) and to present a novel minimally invasive bimanual technique for anterior segment revision surgery associated with ACCS with anterior flexion of the intraocular lens haptics. METHODS A consecutive cohort of 268 eyes of 161 patients undergoing phacoemulsification and implantation of the same type of hydrophilic acrylic aspheric intraocular lens cohort were analysed and a novel technique of minimally invasive bimanual technique for anterior segment revision surgery is described. RESULTS We identified four eyes (1.5%) of three patients with advanced ACCS. Successful restoration of a clear visual axis with minimal induction of astigmatism and rapid visual rehabilitation was achieved in all four cases. CONCLUSION This technique is a safe and minimally invasive alternative to laser or vitrector-cut capsulotomy to restore a clear visual axis. In cases of advanced ACCS, it offers the option for haptic reposition or amputation.

Dynamics of supercooled water in highly compacted clays studied by neutron scattering

The freezing behavior of water confined in compacted charged and uncharged clays (montmorillonite in Na-and Ca-forms, illite in Na-and Ca-forms, kaolinite and pyrophyllite) was investigated by neutron scattering. Firstly, the amount of frozen (immobile) water was measured as a function of temperature at the IN16 backscattering spectrometer, Institute Laue-Langevin (ILL). Water in uncharged, partly hydrophobic (kaolinite) and fully hydrophobic (pyrophyllite) clays exhibited a similar freezing and melting behavior to that of bulk water. In contrast, water in charged clays which are hydrophilic could be significantly supercooled. To observe the water dynamics in these clays, further experiments were performed using quasielastic neutron scattering. At temperatures of 250, 260 and 270 K the diffusive motion of water could still be observed, but with a strong reduction in the water mobility as compared with the values obtained above 273 K. The diffusion coefficients followed a non-Arrhenius temperature dependence well described by the Vogel-Fulcher-Tammann and the fractional power relations. The fits revealed that Na-and Ca-montmorillonite and Ca-illite have similar Vogel-Fulcher-Tammann temperatures (T-VFT, often referred to as the glass transition temperature) of similar to 120 K and similar temperatures at which the water undergoes the 'strong-fragile' transition, T-s similar to 210 K. On the other hand, Na-illite had significantly larger values of T-VFT similar to 180 K and T-s similar to 240 K. Surprisingly, Ca-illite has a similar freezing behavior of water to that of montmorillonites, even though it has a rather different structure. We attribute this to the stronger hydration of Ca ions as compared with the Na ions occurring in the illite clays.

Diversity of morphological patterns in supramolecular polymers formed from the amphiphilic oligomers of pyrenes

Very important aspects of the modern nanotechnology are control and prediction of arraying patterns of opto- and electroactive molecules in discrete objects on nanoscale level both on surface and solution. Consequqntly, a self-assembly of small molucules provides such an opportunity.For example, oligopyrenotides (OPs, short amphiphilic pyrene oligomers) represent a novel class of amphiphilic molecules which tend to aggegate in aqueous phase. As has been already shown, OPs are able to form 1D supramolecular polymer only under high salt concentration. Since programmed arraying of polyaromatic hydrocarbons in structurally defined objects could offer enhanced performance over the individual components, prediction and controlling of their spatial arrangement remains challenging. Herein we demonstrate that substitution type of the pyrene is crutial, and it determines a morphology of the assemblies. Thus, a 1.6-linkage causes a formation of large, free-standing 2D supromolecular polymers with a thickness 2 nm. These assemblies possess a high degree of an internal order: the interior consists of hydrophobic pyrenes and alkyl chains, whereas the exterior exists as a net of hydrophilic negatively charged phosphates. Contrary, a 1.8-linkage exclusiveley leads to a formation of long (up to a few micrometer), nanometer thick helical supramolecular polymers. These structures tend to form even more complex structures (bundles, superhelixes). Moreover for both molecules, the polymerizations occurs via a nucleation-elongation mechanism. To study Py3 self-assembly, we carried out whole set of spectroscopic (UV/vis, fluorescence, DLS) and microscopic experiments (AFM).

Diversity of morphological patterns in supramolecular polymers formed from the amphiphilic oligomers of pyrenes

Herein we demonstrate that a substitution type of the pyrene in short amphiphilic oligomers determines a morphology of the assemblies formed. Thus, 1.6- and 2.7-linkages lead to a formation of micrometer-sized 2D supromolecular polymers with a constant thickness 2 nm (pictures A and B). These assemblies possess a high degree of an internal order: the interior consists of hydrophobic pyrenes and alkyl chains, whereas the exterior exists as a net of hydrophilic negatively charged phosphates. Contrary, a 1.8-linkage exclusiveley leads to a formation of long nanometer thick helical supramolecular polymers (picturee C). These structures tend to form even more complex assemblies (bundles, superhelixes). Moreover, for all samples the polymerization process occurs via a nucleation-elongation mechanism. To study Py3 self-assembly, we carried out whole set of spectroscopic (UV/vis, fluorescence, DLS) and microscopic experiments (AFM).

Experimental peri-implant mucositis at different implant surfaces

OBJECTIVES To histologically and immunologically assess experimental peri-implant mucositis at surface enhanced modified (mod) hydrophilic titanium implants. MATERIALS AND METHODS In a split-mouth design (n = 6 foxhounds), four different implants were inserted on each side of the maxilla: three titanium-zirconium alloy implants (TiZr) with either modSLA (sand-blasted, acid etched and chemically mod), modMA (machined, acid etched and chemically mod), or M (machined) surfaces in the transmucosal portion, and one titanium implant with a machined transmucosal portion (TiM). Experimental mucositis was induced at one randomly assigned side (NPC), whereas the contra-lateral maxillary side received mechanical plaque removal three times per week (PC). At 16 weeks, tissue biopsies were processed for histological (primary outcome: apical extension of the inflammatory cell infiltrate measured from the mucosal margin - PM-aICT) and immunohistochemical (CD68 antigen reactivity) analyses. Peri-implant sulcus fluid was analysed for interleukin (IL)-1β, IL-8, matrix metalloproteinase (MMP)-8 and myeloperoxidase (MPO). RESULTS Mean PM-aICT values varied between 1.86 (TiZrmodSLA) and 3.40 mm (TiM) in the UPC group, and between 0.88 (TiZrmodSLA) and 2.08 mm (TiZrM) in the PC group. Mean CD68, IL-1β, IL-8, MMP-8 and MPO values were equally distributed between mod- and control implants in both NPC and PC groups. CONCLUSIONS The progression of experimental mucositis was comparable at all implant surfaces investigated.

Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters.