Implications of adopting a biodiversity-based vulnerability index versus a shoreline environmental sensitivity index on management and policy planning along coastal areas.

In this study, a multi-criteria index was developed to assess anthropogenic stressors along the Mediterranean coastline. The index aimed at geo-locating pollution hotspots for informed decision making related to coastal zone management. The index was integrated in a Geographical Information System based geodatabase implemented at several pilot areas along the Northern (Italy and France), Eastern (Lebanon), and Southern (Tunisia) Mediterranean coastlines. The generated stressor maps were coupled with a biodiversity richness index and an environmental sensitivity index to produce vulnerability maps that can form the basis for prioritizing management and mitigation interventions towards the identification of pollution hotspots and the promotion of sustainable coastal zone management. The results identified significant differences between the two assessment methods, which can bias prioritization in decision making and policy planning depending on stakeholders' interests. The discrepancies emphasize the need for transparency and understanding of the underlying foundations behind vulnerability indices and mapping development.

Determinants of diarrhea prevalence in urban slums: a comparative assessment towards enhanced environmental management.

This study relies on a comparative assessment of diarrhea occurrence in two urban slums to identify salient factors influencing case prevalence. Primary data were collected from both areas using a structured closed-ended questionnaire coupled with bottled and public water quality sampling and analysis at households reporting diarrhea cases. The water quality analysis showed contamination at the household level due primarily to the location of water storage tanks, as well as in some brands of bottled water due to lack of enforcement of source monitoring. Descriptive statistics and chi-square distribution tests revealed significant difference in diarrhea cases in both study areas which was correlated with the educational level of household head, financial status, type of water storage tank, and corresponding cleaning frequency as well as the adoption of measures to treat water or the use of bottled water.

Public perception and economic implications of bottled water consumption in underprivileged urban areas.

This paper presents a comparative assessment of public perception of drinking water quality in two underprivileged urban areas in Lebanon and Jordan with nearly similar cultural and demographic characteristics. It compares the quality of bottled water to the quality of the drinking water supplied through the public network and examines the economic implications of bottled water consumption in the two study areas. Participants' perception of the quality of drinking water provided via the public network was generally negative, and bottled water was perceived to be of better quality in both areas, thus affecting drinking water preferences and consumption patterns. The results reveal that the quality of bottled water is questionable in areas that lack enforcement of water quality standards, thus adding to the burden of an already disadvantaged community. Both areas demonstrated a considerable cost incurred for purchasing bottled water in low income communities reaching up to 26 % of total income.

ADM1 performance using SS-OFMSW with non-acclimated inoculums.

This paper assesses the anaerobic digestion (AD) of the source-sorted organic fraction of municipal solid waste (SS-OFMSW). For this purpose, an experimental programme was implemented involving the operation and monitoring of two bench-scale anaerobic digesters, continuously fed with SS-OFMSW. The mathematical model (ADM1) was then applied to simulate the process of AD of SS-OFMSW. While start-up of the digesters was relatively slow, re-inoculation with cattle manure with effluent dilution reduced the acclimation period and achieved better stability, accommodating a feeding rate at an OLR = 2.39 kg TVS m(-3) day(-1). The high recorded methane gas production rate, reaching (0.1-2.5 m(3) CH(4)/m(3) reactor day), confirms the excellent biodegradability of the type of waste used (SS-OFMSW) and its suitability for AD. Satisfactory simulations of soluble chemical oxygen demand (COD), pH, and methane composition of biogas were obtained, whereas volatile fatty acid (VFA) concentrations in both reactors were over-predicted albeit capturing its general trend.

HIV-1 integrase and virus and cell DNAs: complex formation and perturbation by inhibitors of integration.

HIV-1 integrase (IN) catalyzes integration of viral DNA into cell DNA through 3'-processing of viral DNA and strand transfer reactions. To learn on binding of IN to DNAs and IN inhibition we applied spectroscopy (circular dichroism, fluorescence) in a simplified model consisting in a peptide analogue (K156) of alpha4 helix involved in recognition of viral and cell DNA; an oligonucleotide corresponding to the U5' LTR DNA end; and an inhibitor (TB11) of the diketo acid (DKA) family. Results extrapolated to IN show that: the enzyme binds viral DNA with high affinity and specificity, but cell DNA with low affinity and specificity; the affinity of TB11 for IN is high enough to impair the binding of IN to cell DNA, but not to viral DNA. This explains why TB11 is an inhibitor of strand transfer but not of 3'-processing. These results can help in the search of new IN inhibitors.

Influence of L-cystinyl side-chain configurations on the melting of crosslinked alpha-tropomyosin dimers.

The experimental melting profile reported by Holtzer, M.E., Holtzer, A. and Skolnick, J. (Macromolecules 16 (1983) 173-180) for the rabbit alpha-tropomyosin dimer crosslinked at cysteine residue 190 has been analyzed using matrix methods. The configuration partition function employed includes a term arising from interactions at the crosslink site. This term, denoted by omega, is found to be smaller than 1, implying that events at the crosslink site resist helix formation by dimer. A theoretical analysis of the conformational restrictions imposed on the crosslink provides a satisfactory estimate of omega at high temperatures. Agreement deteriorates at lower temperatures, perhaps as a consequence of difficulty in establishing a reliable value for omega from analysis of the low-temperature circular dichroism data.

1H and 31P nuclear magnetic resonance studies of the differences in DNA deformation induced by anti-tumoral 7H-pyrido[4,3-c]carbazole dimers.

Ditercalinium (2,2'-[( 4,4'-bipiperidine]-1,1'-diyldi-2,1-ethane-diyl) bis-[10-methoxy-7H pyrido[4,3-c]carbazolium)tetramethane sulfonate (NSC 366241], a DNA bis-intercalating compound, is a potent anti-tumoral rigid dimer. Previous studies have shown that a reduced flexibility of the linking chain of such a dimer is essential for its biological activity. In order to understand, at the molecular level, the mechanism of action and the structure-activity relationships of this series of DNA intercalators, new dimers with additional methylene groups between the two piperidine rings have been synthesized. Addition of one methylene group in the chain preserved the activity, whereas addition of two methylene groups reduced the cytotoxicity, which finally disappeared when three methylene groups were inserted. Therefore, the study of the interaction of dimers bearing no (202), two (222) and three (232) methylene groups with the self-complementary hexanucleotide d(CGATCG)2 have been investigated by 1H and 31P nuclear magnetic resonance studies. The results reported here indicate that all dimers bis-intercalate into the minihelix. The intermolecular nuclear Overhauser effects (NOEs) between the dimers and the nucleotide lead to the conclusion that the three dimers intercalate with their rigid bis-ethyl bipiperidine chain fitting the major groove of the helix. Inter-residue nuclear Overhauser effects at the DNA level, as well as induced shifts, are discussed in relation to the conformational changes induced in DNA upon intercalation and to the different activity of the dimers.

Comparative structural analysis by [1H,31P]-NMR and restrained molecular dynamics of two DNA hairpins from a strong DNA topoisomerase II cleavage site.

The structural analysis of two single-stranded DNAs d(AGCTTATCATCGATAAGCT) (ATC-19) and d(AGCTTATCGATGATAAGCT) (GAT-19) was performed by NMR and restrained molecular dynamics. These oligonucleotides reproduce the 15-33 segment of phage pBR322 DNA, which contains a strong cleavage site for topoisomerase II coupled to the antitumor drugs VP-16 and ellipticine. Because of their partial palindromic nature, the two oligonucleotides ATC-19 and GAT-19 may fold back into stable hairpin structures, consisting of a stem of eight base-pairs and a loop of three residues. NMR assignments and conformational parameters were determined from combined 2D NOESY, COSY and 1H-31P spectra. Conformations of ATC-19 and GAT-19 hairpins were calculated using the X-PLOR 3.1 program. Structures were generated through simulated annealing procedures starting from 50 structures with randomized torsion angles. A good convergence was observed for ATC-19 molecules, while no consensus was found for GAT-19. Within the GAT-19 loop, the base stacking was poor and no hydrogen bond could be detected. In contrast, ATC-19 displayed a well-defined three residue loop stabilized by both extensive base stackings and hydrogen bonding between the N3 atom of the adenine ring and the amino group of the cytosine ring. The results confirm our earlier ATC-19 structure obtained by a completely different calculation procedure (JUMNA) and the higher thermal stability of ATC-19 compared to GAT-19. Moreover, due to its mismatched base-pair, the ATC-19 loop may be better described as a single residue loop rather than a three residue loop. Comparison of this loop to those containing sheared purine.purine base-pairs revealed striking resemblances, particularly on the backbone angle combination. Finally, the differences observed between the ATC-19 and GAT-19 structures could help toward understanding the sequential cleavage of DNA strands by topoisomerase II.

The hairpin structure of a topoisomerase II site DNA strand analyzed by combined NMR and energy minimization methods.

We report on the structural study of the single-stranded 19mer oligonucleotide d(AGCTTATC-ATC-GATAAGCT) 22(+). This corresponds to the 15-to-33(+) strand of pBR322 DNA belonging to a strong cleavage site (site 22) for topoisomerase II coupled to antitumor drugs VP-16 or ellipticine. The partially self-complementary nature of this oligonucleotide makes likely its folding into a hairpin structure. To assess this property we carried out a quantitative analysis based on joint calculations and NMR experiments. The latter required two-dimensional (NOESY, P-COSY, TOCSY and proton-detected 1H-31P), and three-dimensional (NOESY-TOCSY) spectra to achieve the assignment of the overcrowded sugar H4' ad H5'/H5" proton region. For molecular modeling, the JUMNA program was used together with NMR constraints; namely, the distances and the backbone torsion angles provided by NOEs and homo- and heteronuclear coupling constants. Experimental results proved that the 19mer oligonucleotide adopted a stable hairpin structure characterized by an eight base-pair stem and a three-membered loop (central-ATC-segment). Homonuclear 1H-1H and heteronuclear 1H-31P coupling constant measurements provided information on the conformational heterogeneity of the sugar and phosphate groups within both the stem and the loop. Restrained energy minimizations starting with different structures resulted in a family of closely related structures. All low-energy molecules presented the same, rather compact, folded structure with the base-stacking continuing into the loop, a sharp turn occurring between residues T10 and C11, and strong backbone distortions at the loop-stem junction.

Patterns of use of oral anticancer treatments in France: a Retrospective Analysis of Cancer Treatments given ORally from 2004 to 2012 (Re-ACTOR study).

BACKGROUNDS AND OBJECTIVES: Increasing use of oral anticancer treatments (OATs) in oncology is modifying the treatment paradigm for cancer. Nonetheless, available data on the pattern of use of OATs and its evolution over time are limited. The objective of this study was to describe the patterns of use of OATs in France from 2004 to 2012. METHODS: A retrospective analysis was performed using Oncology Analyzer, a physician survey database. All patients actively treated by an oral or an intravenous anticancer treatment between October 2004 and September 2012 were enrolled in the database. Descriptive analyses were performed by treatment category with a focus on the last year of collection and the evolution across the study period. RESULTS: From October 2011 to September 2012, a sample of 7426 patients treated by oral or intravenous active anticancer treatments was analyzed: 74% of patients receiving an OAT were diagnosed with a solid tumor, 52% of whom had a stage IV cancer. The use of OATs increased with age and was the highest in patients over 80 years. From 2004 to 2012, the proportion of cancer patients receiving OATs increased by four percentage points (from 28.4% to 32.5%). Additionally, for treatments available in both forms, a marked preference for oral formulations was observed. LIMITATIONS: The patterns and trend of use prior to 2004 were not addressed due to lack of information in the database. The use of a market research database is relevant for highly prevalent cancers but for rare cancers the sample size is limited, underlining the utility of using other data sources such as cancer registries. CONCLUSIONS: The Re-ACTOR study provides an overview of OAT use in France, which was prescribed to 32% of cancer patients in France in 2012, principally to older patients and to those with solid tumors and with metastatic disease.

Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin.

We report the simultaneous presence of two phospholipase A(2) (PLA(2)) neurotoxins in the venom of Vipera aspis aspis, the first such observation. One is monomeric and identical to ammodytoxin B of Vipera ammodytes ammodytes. Its presence may result from gene flux after interbreeding between V. aspis aspis and V. ammodytes ammodytes. The second, a novel heterodimer named vaspin, is very similar to vipoxin of Vipera ammodytes meridionalis and to PLA(2)-I of Vipera aspis zinnikeri. It may result from expression of preexisting genes, the acidic subunit evolving from an ancestor common to ammodytin I2 from V. ammodytes ammodytes, which we also found in V. aspis aspis.

Synthesis and structure-activity studies of a series of [(hydroxybenzyl)amino]salicylates as inhibitors of EGF receptor-associated tyrosine kinase activity.

The synthesis and structure-activity relationships of a series of [(hydroxybenzylidene)amino]salicylates and a series of [(hydroxybenzyl)amino]salicylates as inhibitors of EGF receptor-associated tyrosine kinase activity are described. Their inhibitory potency was evaluated in vitro using ER 22 cell membranes (CCL 39 cells transfected with EGF receptor) as an enzyme source and the tridecapeptide RRSrc (RRLIEDAEYAARG) as substrate. Their cellular activity was measured by inhibition of the EGF-stimulated DNA synthesis of ER 22 cells. Chemical modifications were made to analyze the role of the different substituents. The amino series was found to be more active than the imino series. The hydroquinone moiety appears to be essential for tyrosine kinase inhibitory activity in the series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Comparison of the imino and amino series by molecular modeling techniques provides further evidence in support of the hypothesis that the important reduced linking chain, CH2NH, allows the correct positioning of the 2,5-dihydroxybenzyl ring, possibly in a cis-like conformational arrangement.

Preferred antagonist binding state of the NMDA receptor: synthesis, pharmacology, and computer modeling of (phosphonomethyl)phenylalanine derivatives.

A series of substituted [phosphono-, sulfo-, carboxy-, and (N-hydroxycarbamoyl)methyl]phenylalanines were synthesized as probes for the investigation of the preferred antagonist state of the NMDA receptor antagonists. The potency of these compounds was evaluated by measuring electrophysiological responses induced by NMDA in cultured mouse cortical neurons. 3-(Phosphonomethyl)phenylalanine [1(m)] a formal AP7 analogue, has been shown to be the most potent antagonist in this study with an IC50 of around 5 microM. The isomeric 2-(phosphonomethyl)phenylalanine [1(o)] was about half as active as 1(m) and as active as compound 5(3), a derivative which is cis-hydrogenated on the phenyl ring of 1(m). Replacement of a phosphono by a sulfo group led to a large reduction in the ability of these compounds to antagonize NMDA responses, although the ortho and meta isomers retained some activity in their reduced forms. In both series the para isomers were almost completely inactive at 100 microM. Introduction of a carboxyl or a bidentate HONHCO group in place of the phosphono moiety in the 3-position results in compounds devoid of activity. The active and inactive compounds of this study were used in conjunction with the most potent linear and cyclic phosphono-containing NMDA antagonists reported to date to determine, via computer modeling techniques, a three-dimensional model corresponding to a antagonist preferring state of the NMDA binding site. This structure defines a pharmacophore which is characterized by (i) well-defined distances between the central atoms of the polar groups PO3H-, NHn+, (n = 2, 3), and COO- (P-N = 5.89 +/- 0.12 A, P-C = 6.66 +/- 0.08 A, and N-C = 2.28 +/- 0.01 A), (ii) a sterically allowed region between the C5 methylene and the PO3H- group, and (iii) a molecular electrostatic field in which the positive, neutral, and negative potential zones are self-contained--with the negative potential zone connecting the PO3H- and COO- groups as the largest. We have compared our results to a preliminary model of the NMDA antagonist site by Hutchison et al. and to a topological model of the NMDA-glycine receptor site by Cordi et al. Our proposed steric-electrostatic pharmacophore which refines, simplifies, and improves these models has now to be validated by the design of new NMDA antagonists.

Induction of DNA bending by bifunctional intercalating agents of the 7H-pyridocarbazole family.

The structures and binding energetics of selected complexes formed between the deoxynucleotides d(CpGpGpCpG).d(CpGpCpCpG), d(CpGpApTpCpG)2, d(GpCpGpCpCpG).d(CpGpGpCpGpC), and d(CpGpCpCpCpG)2 with the DNA bifunctional intercalating agent ditercalinium and three of its rigid linking chain derivatives have been investigated theoretically by means of a molecular mechanics approach that takes into account nucleic acid flexibility, ligand flexibility and solvent dielectric effects (R. Lavery, in: Unusual DNA structures, eds S. Harvey and R. Wells (Pergamon, New York, 1988) p. 189; R. Lavery, in: DNA bending and curvature, eds W.K. Olson et al. (Adenine Press, New York, 1988) p. 191). The piperidinium chains of the bis-intercalating ligands are always located in the major groove of DNA. For the energy-minimized complexes the ligand proceeds to bind following preferentially the 5'-pyrimidine-purine-3' alternating sequence, thus dictating the number of internal exclusion sites. The complexes with three exclusion sites will present (i) a bending of the structure towards the major groove, and (ii) a non-ideal distribution of unwinding angles; complexes with less than three exclusion sites will remain essentially linear. The absence of a bend does not preclude other types of local deformations of the base-pairs such as inclination, buckle and tip. The proposed structures of the d(CpGpApTpCpG)2 complexes are in agreement with NMR structural results. The possible relevance of these findings to a previously proposed mode of interaction for ditercalinium-like DNA ligands is discussed.

Molecular modeling of an active loop structure in lysozyme. Sequence effects or crystal packing?

The Val99-Gly 104 variable region in egg white lysozyme is part of the active site cleft and of the epitope recognized by some monoclonal antibodies. In general, this loop is found in a conformation inflected towards the active site (proximal conformational) such as in free hen lysozyme (HEL). But in a lysozyme such as Japanese quail's (JEL), the loop turns away from the active site cleft (distal conformation). In order to differentiate sequence effects from crystal packing, we generated and refined loop conformations for the 99-104 variable region in lysozyme, then estimated their relative conformational free energies. Some of the results indicate that (i) the flexibility of the 99-104 segment is much greater for HEL than for JEL sequences when unconstrained by the crystal lattice, (ii) for JEL, only distal structures are favored, while for HEL the states span the zone between proximal and distal regions, and (iii) epitopes elucidated from crystal structures may not always be conserved in solution. For the JEL loop, model building shows that an energy-costly distal to proximal transition appears necessary. Finally, analysis of available structural data indicates that changes of humidity, temperature and pressure on loop conformation are negligible.

Intercalative binding of ditercalinium to d(CpGpCpG)2: a theoretical study.

The structure of the complex formed between ditercalinium, 2,2'-[4,4'-bipiperidine-1,1'-bis-(ethane-1,2-diyl)]bis(10-me thoxy-7H- pyrido[4,3-c]carbazolium) tetramethane sulfonate (NSC 366241), and the self-complementary tetranucleotide duplex d(CpGpCpG)2 has been investigated by means of a novel theoretical approach for modelling the conformational flexibility of nucleic acids. The methodology used is the JUMNA procedure, a molecular mechanics systematics capable of evaluating the internal energy and the interaction energy of a complex formed from a large number of fragments. In the best energy-minimized structures, the piperidinium chains of ditercalinium are located in the major groove of the right-handed oligonucleotide. Calculations show a distortion of the base-paired d(CpGpCpG)2 minihelix consisting of lateral dislocation of one base pair with respect to another along an axis parallel to the long axis; strong propeller twist and tilt of the end base pairs; a collective motion of all base pairs with respect to the helical axis towards the drug; and an overwinding at the exclusion site. The proposed structure of the complex is in good agreement with reported proton NMR data, supporting the feasibility of such model.

Molecular basis for the partition of the essential functions of thrombin among snake venom serine proteinases: the case of thrombin-like enzymes.

Thrombin is a mammalian serine proteinase that plays a prominent role in the maintenance and regulation of hemostasis through its interaction with various substrates and/or ligands. The venoms of several snakes contain glycosylated serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen (Fg) and/or platelets. These proteinases share about 60% sequence identity. One class of snake venom serine proteinases are those known as thrombin-like (TLE), named after their ability to directly clot Fg in order to preferentially produce fibrinopeptide A, fibrinopeptide B or both. To understand the molecular basis of this phenomenon, the corresponding amino acid sequences and molecular structures need to be analyzed. Given the absence of experimentally determined tertiary structures of snake venom, TLEs, three-dimensional molecular models should prove useful in this context. Towards this goal, we obtained models of snake venom TLEs that used TSV-PA as template, TSV-PA being the only snake venom serine proteinase whose crystal structure is known to date. Along with a comparative sequence analysis the models contribute to the identification and description of thrombin-homologous or alternative binding sites, helping thus to understand differences in specificity.

A theoretical investigation of the intercalative binding of 7-H pyrido[4.3C]carbazole chromophore into a d(CpG)2 minihelix.

Theoretical computations are performed of the intercalative binding to a model d(CpG)2 minihelix of 7-H pyrido[4.3C]carbazole, the precursor of the antitumor bisintercalating drug ditercalinium. The conformations of the intercalation site are generated by the AGNAS procedure (algorithm to generate nucleic acid structures) of Miller and co-workers. The ligand-nucleotide interactions and the nucleotide conformational energies are computed with the SIBFA procedures (sum of interactions between fragments ab initio computed), which use formulas of empirical origin that reproduce ab initio SCF (self-consistent field) computations. Among the candidate intercalation sites most favored energetically, one has a pattern of conformational angles related to the one determined crystallographically by Sobell et al. in a series of x-ray structural studies of small intercalator-dinucleotide monophosphate complexes. Optimal values of the unwinding angle, found in the range of -12 degrees to -14 degrees, are consistent with available experimental data on DNA.

Identification of the substrate-binding exosites of two snake venom serine proteinases: molecular basis for the partition of two essential functions of thrombin.

The venom of the South American snake Bothrops jararaca contains two serine proteinases, bothrombin and the platelet-aggregating enzyme PA-BJ, which share 66% sequence identity. Each of these proteinases possesses one of the two essential procoagulant functions of thrombin-the clotting of fibrinogen and platelet aggregation. Thus, bothrombin clots fibrinogen but has no direct effect on platelets, unless in the presence of exogenous fibrinogen. PA-BJ induces platelet aggregation by interacting with the protease-activated platelet receptor without clotting fibrinogen. On the other hand, thrombin possesses two extended surfaces. One is composed of basic and hydrophobic residues (exosite I) and the other one of basic residues only (exosite II). These exosites are involved in the recognition of physiological macromolecular substrates. In order to identify the corresponding exosites in bothrombin and PA-BJ and understand the molecular basis of the partition of the two procoagulant functions of thrombin among the two snake venom enzymes, we used molecular modeling to obtain models of their complexes with their natural substrates fibrinogen and a fragment of the protease-activated platelet receptor, respectively. In analogy to thrombin, each of the enzymes presents two exosites. Nonetheless, the exosites contain a smaller proportion of basic residues than thrombin does (45-72%), reducing thus the functional diversity of the enzymes. In addition, the composition of exosite I is different in both enzymes. We identify those residues in exosite I that could contribute to the differences in specificity. Finally, allostery does not seem to mediate macromolecular substrate recognition by these enzymes.

Trimeresurus stejnegeri snake venom plasminogen activator. Site-directed mutagenesis and molecular modeling.

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L., and Bon, C. (1995) J. Biol. Chem. 270, 10246-10255). To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site. When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity. Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the Km for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.