gabriela Gomez

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer's and Parkinson's diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein α-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH2) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of α-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH2 labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH2 labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.

Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis beta-lactamase (BL-betaL) with 1 mM 3H-diazirine yielded 8.3 x 10(-3) mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary structure, a statement supported by the fact that the largest change occurs along the N-I portion of the N-I-U transition and along the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N, an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentation of labeled BL-betaL into peptides provides a sequential map of solvent accessibility. Thus, amino acid residues pertaining to the Omega-loop and to helices alpha5 and alpha6 line the major cavity of the protein, that is big enough to lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique) experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possible a comparative structural characterization of the native as well as non-native states.

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer's and Parkinson's diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein α-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH2) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of α-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH2 labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH2 labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.

Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis beta-lactamase (BL-betaL) with 1 mM 3H-diazirine yielded 8.3 x 10(-3) mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary structure, a statement supported by the fact that the largest change occurs along the N-I portion of the N-I-U transition and along the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N, an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentation of labeled BL-betaL into peptides provides a sequential map of solvent accessibility. Thus, amino acid residues pertaining to the Omega-loop and to helices alpha5 and alpha6 line the major cavity of the protein, that is big enough to lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique) experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possible a comparative structural characterization of the native as well as non-native states.

... informatics study team at St George's — University of London) grants from Roche to process routinely collected general practice data as part of the NEOERICA study.Gabriela Gomez and Hugh Gallagher have no conflicts of interest. ...

Estimated prevalence of self-medication for sexually transmitted diseases (STDs) in observational studies differs between studies and it may vary according to population characteristics. We investigated the extent of self-medication use for STD and explored population and study level factors correlated to the variability observed between populations. Medical databases were systematically searched for published studies. Crude prevalence data were extracted. Pooled estimates were calculated using random effects models. Univariate and multivariate meta-regression models with categorical population level covariates were constructed. Of the 35 studies that met the inclusion criteria for all self-medication use, 20 were also included in the analysis of antibiotic only use. There was significant heterogeneity present across prevalence estimates for all self-medication, Q = 3954.82, P <0.001; and among antibiotic only self-medication, Q = 3797.94, P <0.001. In meta-regression analyses, publication year seemed to explain 22.5% of variation among studies of self-medication with antibiotic only. No other significant associations with population or study characteristics were found. No population level factors leading to high levels of self-medication use for STD were identified. Publications before year 2000 seem to present a higher prevalence of antibiotic use for self-medication. Greater risk awareness of antibiotic misuse and improved control of antibiotic availability at population level might explain this result. An analysis of individual level characteristics should indicate which are influential and whether their local prevalence can explain the observed heterogeneity better.

The landscape of diagnostic testing for tuberculosis (TB) is changing rapidly, and stakeholders need urgent guidance on how to develop, deploy and optimize TB diagnostics in a way that maximizes impact and makes best use of available resources. When decisions must be made with only incomplete or preliminary data available, modelling is a useful tool for providing such guidance. Following a meeting of modelers and other key stakeholders organized by the TB Modelling and Analysis Consortium, we propose a conceptual framework for positioning models of TB diagnostics. We use that framework to describe modelling priorities in four key areas: Xpert(®) MTB/RIF scale-up, target product profiles for novel assays, drug susceptibility testing to support new drug regimens, and the improvement of future TB diagnostic models. If we are to maximize the impact and cost-effectiveness of TB diagnostics, these modelling priorities should figure prominently as targets for future research.

Estimated prevalence of self-medication for sexually transmitted diseases (STDs) in observational studies differs between studies and it may vary according to population characteristics. We investigated the extent of self-medication use for STD and explored population and study level factors correlated to the variability observed between populations. Medical databases were systematically searched for published studies. Crude prevalence data were extracted. Pooled estimates were calculated using random effects models. Univariate and multivariate meta-regression models with categorical population level covariates were constructed. Of the 35 studies that met the inclusion criteria for all self-medication use, 20 were also included in the analysis of antibiotic only use. There was significant heterogeneity present across prevalence estimates for all self-medication, Q = 3954.82, P <0.001; and among antibiotic only self-medication, Q = 3797.94, P <0.001. In meta-regression analyses, publication year seemed to explain 22.5% of variation among studies of self-medication with antibiotic only. No other significant associations with population or study characteristics were found. No population level factors leading to high levels of self-medication use for STD were identified. Publications before year 2000 seem to present a higher prevalence of antibiotic use for self-medication. Greater risk awareness of antibiotic misuse and improved control of antibiotic availability at population level might explain this result. An analysis of individual level characteristics should indicate which are influential and whether their local prevalence can explain the observed heterogeneity better.