Spectral CT and its specific values in the staging of patients with non-small cell lung cancer: technical possibilities and clinical impact.

AIM: To investigate how spectral computed tomography (SCT) values impact the staging of non-small cell lung cancer (NSCLC) patients. MATERIALS AND METHODS: One hundred and thirteen patients with confirmed NSCLC were included in a prospective cohort study. All patients underwent single-phase contrast-enhanced SCT (using the fast tube voltage switching technique, 80-140 kV). SCT values (iodine content [IC], spectral slope pitch, and radiodensity increase) of malignant tissue (primary and metastases) and lymph nodes (LNs) were measured. Adrenal masses were evaluated in a virtual non-contrast series (VNS). If pulmonary embolism was present, pulmonary perfusion was analysed as an additional finding. RESULTS: Fifty-two untreated primary NSCLC lesions were evaluable. Lung adenocarcinoma had significantly higher normalised IC (NIC: 19.37) than squamous cell carcinoma (NIC: 12.03; p=0.035). Pulmonary metastases were not significantly different from benign lung nodules. A total of 126 LNs were analysed and histologically proven metastatic LNs (2.08 mg/ml) had significantly lower IC than benign LNs (2.58 mg/ml; p=0.023). Among 34 adrenal masses, VNS identified adenomas with high sensitivity (91%) and specificity (100%). In two patients, a perfusion defect due to pulmonary embolism was detected in the iodine images. CONCLUSION: SCT may contribute to the differentiation of histological NSCLC subtypes and improve the identification of LN metastases. VNS differentiates adrenal adenoma from metastasis. In case of pulmonary embolism, iodine imaging can visualise associated pulmonary perfusion defects.

Feasibility study of nonclinical safety assessments on homeopathic preparations using the example of protoanemonin in Pulsatilla pratensis L.

Homeopathy is a world-wide available form of complementary therapy, which has a tradition of 200years. Due to the long history of clinical use, i.e. reflected by the first edition of the Homeopathic Pharmacopoeia of the US of 1914, the conduct of toxicological studies is not required if the safety has been otherwise substantiated. The aim of this article is to establish a risk assessment procedure without full toxicological examination, using homeopathic preparations from Pulsatilla pratensis L. as an example. The literature review shows that protoanemonin is the most relevant constituent of these plants regarding potential toxicity. Based on structural alerts protoanemonin is classified as a Cramer class III compound with the threshold of toxicological concern (TTC) of 180µg/day in adults. Neither computer aided toxicology methods (Toxtree and Derek Nexus®) nor a literature search revealed any evidence of genotoxic, carcinogenic or teratogenic potential of protoanemonin. The protoanemonin exposure from a maximum daily dose of a typical homeopathic preparation of P. pratensis L. does not exceed the TTC. The presented method is transparent, reproducible and applicable to other homeopathic substances as a use-case scenario for computational toxicology in order to evaluate an approach for safety assessment of homeopathic medicinal products.

CREB signalling regulates early survival, neuronal gene expression and morphological development in adult subventricular zone neurogenesis.

Neural stem cells in the subventricular zone (SVZ) of the lateral ventricles give rise to new interneurons of the olfactory bulb (OB) throughout life. SVZ/OB neurogenesis is influenced by olfactory network activity, which modulates the survival of new neurons during their integration into the OB network. Previous work suggested that such activity-dependent survival is regulated via the CREB signalling pathway. Curiously, CREB signalling is already active during the early developmental stages of adult SVZ/OB neurogenesis. To investigate the role of cell autonomous CREB signalling during early stages of adult SVZ/OB neurogenesis, we ablated CREB-pathway activity in the SVZ/OB neurogenic lineage using a retroviral strategy. Surprisingly, loss of CREB signalling resulted in increased cell death and loss of expression of the neurogenic transcription factor Pax 6, and of a subset of neuronal proteins in migrating neurons of the RMS. Moreover, post-migratory neurons in the OB displayed impaired dendritic development. These results demonstrate an essential role for CREB signalling in maturation of newborn neurons in the OB and uncover a novel role for CREB signalling in the survival and maintenance of neuronal gene expression during the early stages of SVZ/OB neurogenesis.

Morphology and hemodynamics during vascular regeneration in critically ischemic murine skin studied by intravital microscopy techniques.

BACKGROUND: With the understanding of angiogenesis and arteriogenesis, new theories about the orchestration of these processes have emerged. The aim of this study was to develop an in vivo model that enables visualization of vascular regenerating mechanisms by intravital microscopy techniques in collateral arteriolar flap vascularity. METHODS: A dorsal skin flap (15 × 30 mm) was created in mice and fixed into a skinfold chamber to allow for assessment of morphology and microhemodynamics by intravital fluorescence microscopy (IVFM). Laser scanning confocal microscopy (LSCM) was utilized for three-dimensional reconstruction of the microvascular architecture. RESULTS: Flap tpO(2) was 5.3 ± 0.9 versus 30.5 ± 1.2 mm Hg in controls (p < 0.01). The collateral arterioles in the flap tissue were dilated (29.4 ± 5.3 µm; p < 0.01 vs. controls) and lengthened in a tortuous manner (tortuosity index 1.00 on day 1 vs. 1.35± 0.05 on day 12; p < 0.01). Functional capillary density was increased from 121.00 ± 25 to 170 ± 30 cm/cm(2) (day 12; p < 0.01) as a result of angiogenesis. Morphological evidence of angiogenesis on capillary level and vascular remodeling on arteriolar level could be demonstrated by IVFM and LSCM. CONCLUSIONS: Present intravital microscopy techniques offer unique opportunities to study structural changes and hemodynamic effects of vascular regeneration in this extended axial pattern flap model.

Molecular dynamics simulations of lipid bilayers.

During the past decade, computer simulations of bilayers have moved from the realm of model systems to realistic systems containing tens of phospholipids along with the requisite number of water molecules hydrating the entire molecular assembly. Concomitant with the ability to model larger and larger systems, simulators have also begun to utilize more accurate numerical tools to ensure that the temperature, pressure, simulation timescales, parameter sets and long-range electrostatic interactions of bilayers are correctly accounted for in a typical molecular dynamics simulation. With these tools in hand, work has already begun to define the structure, function and dynamics of bilayer, bilayer/small molecule and bilayer/protein systems. Thus, we have reached an era in which simulators will tackle more and more detailed issues regarding complex bilayer systems.

New developments in applying quantum mechanics to proteins.

Algorithmic improvements of quantum mechanical methodologies have increased our ability to study the electronic structure of fragments of a biomolecule (e.g. an enzyme active site) or entire biomolecules. Three main strategies have emerged as ways in which quantum mechanics can be applied to biomolecules. The supermolecule approach continues to be utilized, but it is slowly being replaced by the so-called coupled quantum mechanical/molecular mechanical methodologies. An exciting new direction is the continued development and application of linear-scaling quantum mechanical approaches to biomolecular systems.

Bimetallic nickel complexes for selective CO2 carbon capture and sequestration.

Herein we report a dinickel azacryptand complex that enables fast, selective, and tight CO2 binding from air. Exploiting the affinity of the cavitand towards azides, CO2 release was observed. Despite the stability of the azido complex, UV irradiation under atmospheric conditions proved to be a suitable pathway for N3(-) replacement by CO2.

The Role of Molecular Dynamics Potential of Mean Force Calculations in the Investigation of Enzyme Catalysis.

The potential of mean force simulations, widely applied in Monte Carlo or molecular dynamics simulations, are useful tools to examine the free energy variation as a function of one or more specific reaction coordinate(s) for a given system. Implementation of the potential of mean force in the simulations of biological processes, such as enzyme catalysis, can help overcome the difficulties of sampling specific regions on the energy landscape and provide useful insights to understand the catalytic mechanism. The potential of mean force simulations usually require many, possibly parallelizable, short simulations instead of a few extremely long simulations and, therefore, are fairly manageable for most research facilities. In this chapter, we provide detailed protocols for applying the potential of mean force simulations to investigate enzymatic mechanisms for several different enzyme systems.

Insights into the function of the zinc hydroxide-Thr199-Glu106 hydrogen bonding network in carbonic anhydrases.

The exact functional role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network in the carbonic anhydrases is unknown. However, from the results of molecular dynamics simulations (MD) we are able to better define its function. From computer graphics analysis and MD simulations on the zinc hydroxide form of human carbonic anhydrase II we find that this interaction forces the hydroxide hydrogen atom to be in a "down" position relative to the deep water-binding pocket. From previous work we have found that this pocket is a high-affinity binding site for CO2. We also note that during the timescale of our simulation (126 ps) the hydrogen bonds between the hydroxide hydrogen atom and Thr199 and the one between Thr199 and Glu106 are not fluxional. We propose that the role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network is to lock the hydrogen atom in the down position in order to expose the CO2 molecule bound in the deep water pocket to a lone pair of the hydroxide oxygen atom. This would allow for the rapid reaction of the CO2 molecule around the zinc ion. Furthermore, if the hydroxide hydrogen atom were not locked in the down position the binding of CO2 to the deep water pocket could be interfered with by the unrestrained hydroxide hydrogen atom (e.g. the N-Zn-O-H torsion could undergo rotational transitions that would partially block the deep water pocket). In summary, the roles we ascribe to this hydrogen bonding network are (1) to allow for facile access of CO2 to the deep water pocket and (2) to allow for maximal exposure of a hydroxide oxygen lone pair to the CO2 carbon atom.

One-dimensional molecular representations and similarity calculations: methodology and validation.

Drug discovery research is increasingly dedicated to biological screening on a massive scale, which seems to imply a basic rejection of many computer-assisted techniques originally designed to add rationality to the early stages of discovery. While ever-faster and more clever 3D methodologies continue to be developed and rejected as alternatives to indiscriminant screening, simpler tools based on 2D structure have carved a stable niche in the high-throughput paradigm of drug discovery. Their staying power is due in no small part to simplicity, ease of use, and demonstrated ability to explain structure-activity data. This observation led us to wonder whether an even simpler view of structure might offer an advantage over existing 2D and 3D methods. Accordingly, we introduce 1D representations of chemical structure, which are generated by collapsing a 3D molecular model or a 2D chemical graph onto a single coordinate of atomic positions. Atoms along this coordinate are differentiated according to elemental type, hybridization, and connectivity. By aligning 1D representations to match up identical atom types, a measure of overall structural similarity is afforded. In extensive structure-activity validation tests, 1D similarities consistently outperform both Daylight 2D fingerprints and Cerius(2) pharmacophore fingerprints, suggesting that this new, simple means of representing and comparing structures may offer a significant advantage over existing tried-and-true methods.

Binding preferences of hydroxamate inhibitors of the matrix metalloproteinase human fibroblast collagenase.

In this paper we report molecular dynamics (MD) and free energy perturbation (FEP) studies carried out on enzyme-inhibitor (two hydroxamates that only differ by a carbon-carbon double bond) complexes of human fibroblast collagenase to obtain insights into the structural and energetic preferences of these inhibitors. We have developed a bonded model for the catalytic and structural zinc centers (Hoops, S. C.; et al. J. Am. Chem. Soc. 1991, 113, 8262-8270) where the electrostatic representation for this model was derived using a novel quantum-mechanical/molecular-mechanical (QM/MM) minimization procedure followed by electrostatic potential fitting. The resulting bonded model for the zinc ions was then used to generate MD trajectories for structural analysis and FEP studies. This model has satisfactorily reproduced the structural features of the active site, and furthermore, the FEP simulations gave relative free energies of binding in good agreement with experimental results. MD simulations in conjunction with the FEP are able to provide a structural explanation regarding why one hydroxamate inhibitor is favored over the other, and we are also able to make predictions about changes in the inhibitor that would enhance protein-inhibitor interactions.

Molecular dynamics study of the IIA binding site in human serum albumin: influence of the protonation state of Lys195 and Lys199.

The IIA binding site of human serum albumin (HSA) preferentially binds hydrophobic organic anions of medium size (e.g., aspirin, benzylpenicillin, warfarin, etc.) and bilirubin. This binding ability is particularly important for the distribution, metabolism, and efficacy of drugs. In addition, HSA can also covalently link to different IIA substrates owing to the presence of a highly reactive residue, Lys199, which is strategically located in the IIA site. Herein, we present results of three restrained molecular dynamics (MD) simulations of the IIA binding site on the HSA protein. From these simulations, we have determined the influence that the ionization state of the key residue, Lys199, and the nearby Lys195 has on the structure and dynamics of the IIA binding site. When Lys199 is neutral the computed average distances for the most significant interresidue contacts are in good agreement with those estimated from the X-ray coordinates. The analysis of the solvent structure and dynamics indicates that the basic form of Lys199 is likely connected to the acid form of Lys195 through a network of H-bonding water molecules with a donor --> acceptor character. The presence of these water bridges can be important for stabilizing the configuration of the IIA binding site and/or promoting a potential Lys195 --> Lys199 proton-transfer process. These results suggest that both lysine residues located in the IIA binding site of HSA, Lys195 and Lys199, could play a combined and comparable chemical role. Our simulations also give insight into the binding of bilirubin to HSA.

Prediction of drug absorption using multivariate statistics.

Literature data on compounds both well- and poorly-absorbed in humans were used to build a statistical pattern recognition model of passive intestinal absorption. Robust outlier detection was utilized to analyze the well-absorbed compounds, some of which were intermingled with the poorly-absorbed compounds in the model space. Outliers were identified as being actively transported. The descriptors chosen for inclusion in the model were PSA and AlogP98, based on consideration of the physical processes involved in membrane permeability and the interrelationships and redundancies between available descriptors. These descriptors are quite straightforward for a medicinal chemist to interpret, enhancing the utility of the model. Molecular weight, while often used in passive absorption models, was shown to be superfluous, as it is already a component of both PSA and AlogP98. Extensive validation of the model on hundreds of known orally delivered drugs, "drug-like" molecules, and Pharmacopeia, Inc. compounds, which had been assayed for Caco-2 cell permeability, demonstrated a good rate of successful predictions (74-92%, depending on the dataset and exact criterion used).

Application of the free energy perturbation method to human carbonic anhydrase II inhibitors.

An analysis of the free energy perturbation (FEP) method is presented that attempts to evaluate the efficacy of the FEP method in the drug discovery process. To accomplish this we have evaluated whether the FEP technique can accurately predict energetic and structural quantities relating to the inhibition of human carbonic anhydrase II (HCAII) by sulfonamides. Three well-characterized (both structurally and energetically) sulfonamide inhibitors of HCAII were examined in this study, 1a, 1b, and 1c. Results from FEP simulations on these compounds indicate that the FEP method can predict energetic trends reasonably well; however, the FEP method was less successful in reproducing detailed structural data. In particular, an expected movement of His-64 when inhibitor 1c was bound did not occur. We conclude that the FEP method can be used to determine relative free energies of binding but cannot be relied upon to reproduce subtle geometric changes.

Synthesis of 7-benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine and novel derivatives free of positional isomers. Potent inhibitors of cAMP-specific phosphodiesterase and of malignant tumor cell growth.

7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine (7a) is a potent inhibitor of the cAMP-specific phosphodiesterase isoenzyme family PDE4 and induces growth inhibition in a panel of tumor cell lines. In this study, we describe a synthesis that yields 7a and novel derivatives free of positional isomers. The synthesis of alkylamino substituted pteridines is based on the successive nucleophilic aromatic substitution of the chlorine atoms of 2,4,6, 7-tetrachloropteridine. For the reaction with secondary amines, the positional order of reactivity was found to be C4 > C7 > C2 > C6. Final structural proof is given by X-ray crystallography. To unravel structural elements of 7a crucial for the interaction with the target enzyme, the compound was modified systematically. The impact of the modifications on activity was tested by evaluating the ability of the compounds to inhibit cAMP hydrolysis by cAMP-specific phosphodiesterase (PDE4) purified from the solid human large cell lung tumor xenograft LXFL529. Growth inhibitory properties were determined by in vitro treatment of the respective cell line LXFL529L using the sulforhodamine B assay (SRB). The results show that for high activity, the heterocyclic substituent in position 2 of the pteridine ring system requires the presence of a basic nitrogen in 4'-position, as represented by piperazine.

GB/SA water model for the Merck molecular force field (MMFF).

A revised generalized Born/surface area (GB/SA) continuum solvation model has been developed for water that is compatible with the Merck molecular force field (MMFF). This model gives free energies of aqueous solvation that are comparable in accuracy to the original water model when the OPLS* force field is employed. The average unsigned error in aqueous deltaGsol using the new water model and MMFF is 0.62 kcal/mol for a training set of 82 solutes compared to 1.24 kcal/mol for the original GB/SA water model and MMFF. The average unsigned errors for 47 neutral solutes outside the training set and 10 ions are 0.96 and 2.32 kcal/mol, respectively. By comparison, the average errors for the test set and ions using the original GB/SA water model are 1.76 and 5.32 kcal/mol. This revised parameter set provides a more accurate representation of aqueous solvation for use with MMFF.

Computational prediction of the three-dimensional structures for the Caenorhabditis elegans tubulin family.

In this article we characterize, from a structural point of view, all 16 members of the tubulin gene family of Caenorhabditis elegans (9 alpha-tubulins, 6 beta-tubulins, and 1 gamma-tubulin). We obtained their tertiary structures by computationally modifying the X-ray crystal structure of the pig brain alpha/beta-tubulin dimer published by Nogales et al. [Nature (London) 1998;391:199-203]. Our computational protocol involves changing the amino acids (with MIDAS; Jarvis et al., UCSF MIDAS. University of California, San Francisco, 1986) in the 3D structure of pig brain alpha/beta-tubulin dimer followed by geometry optimization with the AMBER force field (Perlman et al., AMBER 4. University of California, San Francisco, 1990). We subsequently analyze and compare the resulting structures in terms of the differences in their secondary and tertiary structures. In addition, we compare the pattern of hydrogen bonds and hydrophobic contacts in the guanosine triphosphate (GTP)-binding site for all members of the tubulin family. Our computational results show that, except for gamma-tubulin, all members of the C. elegans tubulin family have similar secondary and 3D structures and that the change in the pattern of hydrogen bonds in the GTP-binding site may be used to assess the relative stability of different alpha/beta-tubulin dimers formed by monomers of the tubulin family.

Incorporation of vitamin D3-derivatives in liposomes of different lipid types.

Vitamin D3-derivatives are known to be effective in differentiation and proliferation of epidermal cells. However, under certain circumstances, they also may show a hypercalcamic activity which can be a serious limitation in their use for dermatological application. For keeping small the negative side effects of vitamin D3-derivatives and for increasing the drug concentration in the skin we investigated their incorporation in liposomes to optimize their use for psoriasis treatment. The incorporation of vitamin D3-derivatives in liposomes of different lipid composition was studied by HPLC, DSC and freeze-fracture electron microscopy. Incorporation rates of more than 80% of the offered drug were found with significant variations related to the number of hydroxyl-groups in the A-ring, opened or closed B-ring, and some modifications in the side chain of the steroid-molecules. In general, the incorporation rates in egg-PC liposomes have been of about 3% up to 10% higher than in DMPC liposomes. Results, obtained by DSC and freeze-fracture electron microscopy, show a depression of the phase transition of DMPC bilayers by incorporation of vitamin D3 already at a concentration of 10 mol%. These results support the idea that vitamin D3 and its analoga investigated are incorporated into the lipid bilayer modifying there the lipid-lipid interactions.

Interaction of liposomal incorporated vitamin D3-analogues and human keratinocytes.

The influence of different liposomal qualities, loaded with a variety of vitamin D3-analogues, on the proliferation and interleucine 1 alpha-release (IL-1 alpha) of human keratinocytes was examined by fluorimetric and colorimetric measurements to optimize their use for psoriasis treatment. In comparison, the effects of the free drugs, as 25-hydroxyvitamin D3, calcipotriol, and calcitriol, as well as of empty liposomes have been studied. At the interaction between empty liposomes (< 200 nm) and HaCaT-cells has been looked by electron microscopy. Empty liposomes, made of DMPC as well as of egg-PC, can be used as drug carrier without any inhibiting effect on the proliferation of human keratinocytes at lipid concentrations of < 10(-4) M. Under the influence of the free drugs investigated an inhibition of cell growth as well as of the IL 1 alpha-release was measured at drug concentrations of > or = 10(-8) M. In comparison the related liposomal drug formulations didn't show any diminishing in the proliferation effects caused by the free drugs. A significant improvement, however, was only found in the action of DMPC-incorporated 25-hydroxyvitamin D3 at drug concentration of 10(-7) M. These results suggest that there is no remarkable improvement in the action of liposomal incorporated vitamin D3-analogues neither related to their proliferation nor their IL1 alpha-releasing effects. The influence of liposomal incorporated vitamin D3-analogues in keeping small their negative side effects has to be investigated at a more relevant model.

Lithiated Clusters from Primary Silylphosphanes and Silylarsanes: Spherical Bodies with and without Li(2)O Filling.

Molecular clusters with archimedean and platonic shape, which in order to build a closed polyhedron, spontaneously eliminate H(2) or voluntarily encapsulate Li(2)O as a "cluster nucleus", result from the dilithiation of primary silylphosphanes and silylarsanes with BuLi. Thus, the first mixed-valent, decameric P(10)Li(16) cluster 1 was obtained from iPr(3)SiPH(2) and tBuLi (molar ratio 1:2) with strict exclusion of LiOH and Li(2)O, whereas partial metalation in the presence of LiOH initially leads to a dodecameric, Li(2)O-containing cluster 2, from which the three-shell cluster 3 with a [Li(6)O](4+) core is obtained.