Threats to Validity in the Design and Conduct of Preclinical Efficacy Studies: A Systematic Review of Guidelines for In Vivo Animal Experiments

Background

The vast majority of medical interventions introduced into clinical development prove unsafe or ineffective. One prominent explanation for the dismal success rate is flawed preclinical research. We conducted a systematic review of preclinical research guidelines and organized recommendations according to the type of validity threat (internal, construct, or external) or programmatic research activity they primarily address.

Methods and Findings

We searched MEDLINE, Google Scholar, Google, and the EQUATOR Network website for all preclinical guideline documents published up to April 9, 2013 that addressed the design and conduct of in vivo animal experiments aimed at supporting clinical translation. To be eligible, documents had to provide guidance on the design or execution of preclinical animal experiments and represent the aggregated consensus of four or more investigators. Data from included guidelines were independently extracted by two individuals for discrete recommendations on the design and implementation of preclinical efficacy studies. These recommendations were then organized according to the type of validity threat they addressed. A total of 2,029 citations were identified through our search strategy. From these, we identified 26 guidelines that met our eligibility criteria—most of which were directed at neurological or cerebrovascular drug development. Together, these guidelines offered 55 different recommendations. Some of the most common recommendations included performance of a power calculation to determine sample size, randomized treatment allocation, and characterization of disease phenotype in the animal model prior to experimentation.

Conclusions

By identifying the most recurrent recommendations among preclinical guidelines, we provide a starting point for developing preclinical guidelines in other disease domains. We also provide a basis for the study and evaluation of preclinical research practice. Please see later in the article for the Editors'' Summary     

Assessing “Dangerous Climate Change”: Required Reduction of Carbon Emissions to Protect Young People,Future Generations and Nature

We assess climate impacts of global warming using ongoing observations and paleoclimate data. We use Earth’s measured energy imbalance, paleoclimate data, and simple representations of the global carbon cycle and temperature to define emission reductions needed to stabilize climate and avoid potentially disastrous impacts on today’s young people, future generations, and nature. A cumulative industrial-era limit of ∼500 GtC fossil fuel emissions and 100 GtC storage in the biosphere and soil would keep climate close to the Holocene range to which humanity and other species are adapted. Cumulative emissions of ∼1000 GtC, sometimes associated with 2°C global warming, would spur “slow” feedbacks and eventual warming of 3–4°C with disastrous consequences. Rapid emissions reduction is required to restore Earth’s energy balance and avoid ocean heat uptake that would practically guarantee irreversible effects. Continuation of high fossil fuel emissions, given current knowledge of the consequences, would be an act of extraordinary witting intergenerational injustice. Responsible policymaking requires a rising price on carbon emissions that would preclude emissions from most remaining coal and unconventional fossil fuels and phase down emissions from conventional fossil fuels.     

Energy Expenditure Compared to Physical Activity Measured by Accelerometry and Self-Report in Adolescents: A Validation Study

Background

Physical inactivity is responsible for 5.3 million deaths annually worldwide. To measure physical activity energy expenditure, the doubly labeled water (DLW) method is the gold standard. However, questionnaires and accelerometry are more widely used. We compared physical activity measured by accelerometer and questionnaire against total (TEE) and physical activity energy expenditure (PAEE) estimated by DLW.

Methods

TEE, PAEE (TEE minus resting energy expenditure) and body composition were measured using the DLW technique in 25 adolescents (16 girls) aged 13 years living in Pelotas, Brazil. Physical activity was assessed using the Actigraph accelerometer and by self-report. Physical activity data from accelerometry and self-report were tested against energy expenditure data derived from the DLW method. Further, tests were done to assess the ability of moderate-to-vigorous intensity physical activity (MVPA) to predict variability in TEE and to what extent adjustment for fat and fat-free mass predicted the variability in TEE.

Results

TEE varied from 1,265 to 4,143 kcal/day. It was positively correlated with physical activity (counts) estimated by accelerometry (rho  = 0.57; p = 0.003) and with minutes per week of physical activity by questionnaire (rho  = 0.41; p = 0.04). An increase of 10 minutes per day in moderate-to-vigorous intensity physical activity (MVPA) relates to an increase in TEE of 141 kcal/day. PAEE was positively correlated with accelerometry (rho  = 0.64; p = 0.007), but not with minutes per week of physical activity estimated by questionnaire (rho  = 0.30; p = 0.15). Physical activity by accelerometry explained 31% of the vssariability in TEE. By incorporating fat and fat-free mass in the model, we were able to explain 58% of the variability in TEE.

Conclusion

Objectively measured physical activity significantly contributes to the explained variance in both TEE and PAEE in Brazilian youth. Independently, body composition also explains variance in TEE, and should ideally be taken into account when using accelerometry to predict energy expenditure values.     

Enterococcal and streptococcal resistance to PC190723 and related compounds: Molecular insights from a FtsZ mutational analysis

New antibiotics with novel mechanisms of action are urgently needed to overcome the growing bacterial resistance problem faced by clinicians today. PC190723 and related compounds represent a promising new class of antibacterial compounds that target the essential bacterial cell division protein FtsZ. While this family of compounds exhibits potent antistaphylococcal activity, they have poor activity against enterococci and streptococci. The studies described herein are aimed at investigating the molecular basis of the enterococcal and streptococcal resistance to this family of compounds. We show that the poor activity of the compounds against enterococci and streptococci correlates with a correspondingly weak impact of the compounds on the self-polymerization of the FtsZ proteins from those bacteria. In addition, computational and mutational studies identify two key FtsZ residues (E34 and R308) as being important determinants of enterococcal and streptococcal resistance to the PC190723-type class of compounds.     

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.     

Attempted Integration of Multiple Species of Turaco Into a Mixed‐Species Aviary

Mixed‐species exhibits offer a variety of benefits but can be challenging to maintain due to difficulty in managing interspecific interactions. This is particularly true when little has been documented on the behavior of the species being mixed. This was the case when we attempted to house three species of turaco (family: Musophagidae) together with other species in a walk‐through aviary. To learn more about the behavior of great blue turacos, violaceous turacos, and white‐bellied gray go‐away birds, we supplemented opportunistic keeper observations with systematic data collection on their behavior, location, distance from other birds, and visibility to visitors. Keepers reported high levels of aggression among turacos, usually initiated by a go‐away bird or a violaceous turaco. Most aggression occurred during feedings or when pairs were defending nest sites. Attempts to reduce aggression by temporarily removing birds to holding areas and reintroducing them days later were ineffective. Systematic data collection revealed increased social behavior, including aggression, during breeding season in the violaceous turacos, as well as greater location fidelity. These behavioral cues may be useful in predicting breeding behavior in the future. Ultimately, we were only able to house three species of turaco together for a short time, and prohibitively high levels of conflict occurred when pairs were breeding. We conclude that mixing these three turaco species is challenging and may not be the most appropriate housing situation for them, particularly during breeding season. However, changes in turaco species composition, sex composition, or exhibit design may result in more compatible mixed‐turaco species groups. Zoo Biol. 32:216–221, 2013. © 2012 Wiley Periodicals, Inc.     

Significance of the Natural Occurrence of L‐ Versus D‐Pipecolic Acid: A Review

Pipecolic acid naturally occurs in microorganisms, plants, and animals, where it plays many roles, including the interactions between these organisms, and is a key constituent of many natural and synthetic bioactive molecules. This article provides a review of current knowledge on the natural occurrence of pipecolic acid and the known and potential significance of its L‐ and D‐enantiomers in different scientific disciplines. Knowledge gaps with perspectives for future research identified within this article include the roles of the L‐ versus the D‐enantiomer of pipecolic acid in plant resistance, nutrient acquisition, and decontamination of polluted soils, as well as rhizosphere ecology and medical issues. Chirality 25:823–831, 2013. © 2013 Wiley Periodicals, Inc.