Drugging the undruggable: gabapentin,pregabalin and the calcium channel α2δ subunit

In the post-genomic era, the idea of using the sequence of a protein to determine its potential role as a drug target has gained currency. The goal of this approach to drug discovery is to use the sequence of a protein that is known to bind a specific ligand or drug, along with the known structure of the ligand binding site, to predict other similar proteins that are also “druggable”. Gabapentin (Neurontin) and pregabalin (Lyrica) are drugs currently in the clinic that were developed based on the hypothesis that generating non-hydrolyzable analogs of GABA would lead to the development of antiepileptic agents. While these compounds are indeed good anticonvulsants, their activity is surprisingly not due to activity in the GABAergic system. By purifying the protein to which gabapentin bound, and determining its identity as the α₂δ₁ subunit of voltage gated calcium channels, it was possible to make progress in developing new compounds with similar activities to gabapentin, including pregabalin. The recognition of the α₂δ₁ subunit as the receptor for these drugs also meant that related proteins, such as α₂δ₃, may be interesting targets for novel pain therapeutics.     

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.     

Detoxification of olefinic epoxides and nucleotide excision repair of epoxide-mediated DNA damage: Insights from animal models examining human sensitivity to 1,3-butadiene

1,3-Butadiene (BD) is a well-documented mutagen and carcinogen in rodents and is currently classified as a probable carcinogen in humans. Studies investigating workers exposed to BD indicate that, in some plants, there may be an increased genetic risk, and that polymorphisms in biotransformation and DNA repair proteins may modulate genetic susceptibility. To investigate the role of genetic polymorphisms in microsomal epoxide hydrolase (mEH) or nucleotide excision repair (NER) in contributing to the mutagenicity of BD, we conducted a series of experiments in which mice lacking mEH or NER activity were exposed to BD by inhalation or to the reactive epoxide metabolites of BD (epoxybutene-EB or diepoxybutane-DEB) by i.p. injection. Genetic susceptibility was measured using the Hprt cloning assay. Both deficient strains of mouse were significantly more sensitive to the mutagenic effects of BD and the injected epoxides. These studies provide support for the critical role that mEH plays in the biotransformation of BD, and the role that NER plays in maintaining genomic integrity following exposure to BD. Additional studies are needed to examine the importance of base excision repair (BER) in maintaining genomic integrity, the differential formation of DNA and protein adducts in deficient strains, and the potential for enhanced sensitivity to BD genotoxicity in mice either lacking or deficient in both biotransformation and DNA repair activity.     

Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora

New mutations are found among approximately 20% of progeny when one or both parents carry eas allele UCLA191 (eas(UCLA), easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa3 deficient, linkage group VII), resulting in thin, "transparent" mycelial growth. Other eas alleles fail to produce cya-8 mutant progeny. The recurrent cya-8 mutations are attributed to repeat-induced point mutation (RIP) resulting from a duplicated copy of cya-8+ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas(UCLA)-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas(UCLA), the new mutations arise exclusively in eas(UCLA) nuclei. Mutation of cya-8 is suppressed when a long unlinked duplication is present. Stable cya-8 mutations are effectively eliminated in crosses homozygous for rid, a recessive suppressor of RIP. The eas(UCLA) allele is associated with a long paracentric inversion. A discontinuity is present in eas(UCLA) DNA. The eas promoter is methylated in cya-8 progeny of eas(UCLA), presumably by the spreading of methylation beyond the adjoining RIP-inactivated duplication. These findings support a model in which an ectopic insertion that created a mutation at the target site acts as a locus-specific mutator via RIP.     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Parent overweight predicts daughters' increase in BMI and disinhibited overeating from 5 to 13 years

Objective: To assess whether parental overweight status and disinhibited overeating are predictive of daughters’ accelerated weight gain and disinhibited overeating. Research Methods and Procedures: Participants were part of a longitudinal study of girls (N = 197) and their parents. Measured height and weight were used to calculate BMI [weight (kilograms)/height (meters)²]. Parents’ disinhibited eating behavior was assessed using the Eating Inventory. Girls’ disinhibited eating was assessed using a behavioral protocol to measure eating in the absence of hunger. Girls were classified based on parental overweight at study entry into four groups: neither, mother only, father only, or both parents overweight. Results: Girls with both parents overweight had the most rapid increases in BMI from 5 to 13 years of age; BMI increased most slowly among the neither parent overweight group, with intermediate increases in BMI among mother only and father only overweight groups. Daughters with both parents overweight at study entry were eight times more likely to be overweight at age 13, controlling for daughters’ weight at age 5. Girls with both parents overweight had higher levels of disinhibited eating across all ages than all other groups. Although girls in all parental weight status groups showed increases in disinhibited eating over time, girls with both parents overweight had larger increases in disinhibited eating over time compared with all other groups. Discussion: Girls growing up in families differing in parental overweight had divergent developmental trajectories for BMI and disinhibited overeating. Findings reveal the need to focus prevention efforts on overweight parents of young children.     

Long-Term Carriage of Medicopsis romeroi,an Agent of Black-Grain Mycetoma,Presenting as Phaeohyphomycosis in a Renal Transplant Patient

Mycopathologia - Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a...     

Prevention of violence against women and girls: A cost-effectiveness study across 6 low- and middle-income countries

BackgroundViolence against women and girls (VAWG) is a human rights violation with social, economic, and health consequences for survivors, perpetrators, and society. Robust evidence on economic, social, and health impact, plus the cost of delivery of VAWG prevention, is critical to making the case for investment, particularly in low- and middle-income countries (LMICs) where health sector resources are highly constrained. We report on the costs and health impact of VAWG prevention in 6 countries.Methods and findingsWe conducted a trial-based cost-effectiveness analysis of VAWG prevention interventions using primary data from 5 randomised controlled trials (RCTs) in sub-Saharan Africa and 1 in South Asia. We evaluated 2 school-based interventions aimed at adolescents (11 to 14 years old) and 2 workshop-based (small group or one to one) interventions, 1 community-based intervention, and 1 combined small group and community-based programme all aimed at adult men and women (18+ years old). All interventions were delivered between 2015 and 2018 and were compared to a do-nothing scenario, except for one of the school-based interventions (government-mandated programme) and for the combined intervention (access to financial services in small groups). We computed the health burden from VAWG with disability-adjusted life year (DALY). We estimated per capita DALYs averted using statistical models that reflect each trial’s design and any baseline imbalances. We report cost-effectiveness as cost per DALY averted and characterise uncertainty in the estimates with probabilistic sensitivity analysis (PSA) and cost-effectiveness acceptability curves (CEACs), which show the probability of cost-effectiveness at different thresholds. We report a subgroup analysis of the small group component of the combined intervention and no other subgroup analysis. We also report an impact inventory to illustrate interventions’ socioeconomic impact beyond health. We use a 3% discount rate for investment costs and a 1-year time horizon, assuming no effects post the intervention period. From a health sector perspective, the cost per DALY averted varies between US$222 (2018), for an established gender attitudes and harmful social norms change community-based intervention in Ghana, to US$17,548 (2018) for a livelihoods intervention in South Africa. Taking a societal perspective and including wider economic impact improves the cost-effectiveness of some interventions but reduces others. For example, interventions with positive economic impacts, often those with explicit economic goals, offset implementation costs and achieve more favourable cost-effectiveness ratios. Results are robust to sensitivity analyses. Our DALYs include a subset of the health consequences of VAWG exposure; we assume no mortality impact from any of the health consequences included in the DALYs calculations. In both cases, we may be underestimating overall health impact. We also do not report on participants’ health costs.ConclusionsWe demonstrate that investment in established community-based VAWG prevention interventions can improve population health in LMICs, even within highly constrained health budgets. However, several VAWG prevention interventions require further modification to achieve affordability and cost-effectiveness at scale. Broadening the range of social, health, and economic outcomes captured in future cost-effectiveness assessments remains critical to justifying the investment urgently required to prevent VAWG globally.

In a cost-effectiveness study, Dr. Giulia Ferrari and colleagues examine the costs and health impact of prevention of violence against women and girls in six low- and middle-income countries.     

Culture of the 'aquatic chicken': present concerns and future prospects

This is not actually a swimming chicken, but rather a fish that fulfils a similar role for subsistence farmers and can also be farmed on a commercial scale. Like the chicken, the tilapiines are meeting protein needs in an affordable way world-wide. However, present farming strategies are struggling to meet ever-rising consumer demand.     

Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines.