Mutations designed to destabilize the receptor-bound conformation increase MICA-NKG2D association rate and affinity

MICA is a major histocompatibility complex-like protein that undergoes a structural transition from disorder to order upon binding its immunoreceptor, NKG2D. We redesigned the disordered region of MICA with RosettaDesign to increase NKG2D binding. Mutations that stabilize this region were expected to increase association kinetics without changing dissociation kinetics, increase affinity of interaction, and reduce entropy loss upon binding. MICA mutants were stable in solution, and they were amenable to surface plasmon resonance evaluation of NKG2D binding kinetics and thermodynamics. Several MICA mutants bound NKG2D with enhanced affinity, kinetic changes were primarily observed during association, and thermodynamic changes in entropy were as expected. However, none of the 15 combinations of mutations predicted to stabilize the receptor-bound MICA conformation enhanced NKG2D affinity, whereas all 10 mutants predicted to be destabilized bound NKG2D with increased on-rates. Five of these had affinities enhanced by 0.9-1.8 kcal/mol over wild type by one to three non-contacting substitutions. Therefore, in this case, mutations designed to mildly destabilize a protein enhanced association and affinity.     

Monitoring demographics of a commercially exploited population of shovelnose sturgeon in the Wabash River,Illinois/Indiana,USA

Shovelnose sturgeon (Scaphirhynchus platorynchus, Rafinesque, 1820) in the Wabash River, Illinois/Indiana, USA, provide an important recreational sport and commercial caviar fishery. In fact, it is one of the last commercially viable populations for sturgeon roe harvest. Due to increased demand in the caviar trade and endangered species legislation that protect shovelnose sturgeon in only a portion of their range, efforts of the roe harvest market may continue to divert toward unprotected populations like the shovelnose sturgeon in the Wabash River. Previous studies have shown that increased harvest pressure in this species can affect the age‐at‐maturation and result in recruitment overfishing. Therefore, it is important to closely and continuously monitor commercially exploited populations. Over the past decade (2007–2016), 13,170 shovelnose sturgeon were sampled with boat electroshocking, hoop nets, drift nets, trotlines, and benthic electrified trawls. Captured fish ranged from 61 to 910 mm fork length (FL; mean = 668 mm), with very few fish less than 550 mm FL. Although fish were found to be in a healthy condition (mean relative weight = 87), there was a decrease in the mean condition over time. In addition, we saw declines in mean FL, weight of roe‐per‐fish, and size‐at‐maturity for female fish directly impacted by harvest. The decline of these population parameters, coupled with an increase in total annual mortality and a truncated age frequency distribution, suggest that harvest is negatively impacting the demographics and recruitment of shovelnose sturgeon in the Wabash River. Considering the downward trajectory of population dynamics and high estimates of mortality, their resiliency to continued harvest and environmental changes will be limited.     

Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.     

Validation of a new method for testing provider clinical quality in rural settings in low- and middle-income countries: the observed simulated patient

Background

Assessing the quality of care provided by individual health practitioners is critical to identifying possible risks to the health of the public. However, existing assessment methods can be inaccurate, expensive, or infeasible in many developing country settings, particularly in rural areas and especially for children. Following an assessment of the strengths and weaknesses of the existing methods for provider assessment, we developed a synthesis method combining components of direct observation, clinical vignettes, and medical mannequins which we have termed “Observed Simulated Patient” or OSP. An OSP assessment involves a trained actor playing the role of a ‘mother’, a life-size doll representing a 5-year old boy, and a trained observer. The provider being assessed was informed in advance of the role-playing, and told to conduct the diagnosis and treatment as he normally would while verbally describing the examinations.

Methodology/Principal Findings

We tested the validity of OSP by conducting parallel scoring of medical providers in Myanmar, assessing the quality of their diagnosis and treatment of pediatric malaria, first by direct observation of true patients and second by OSP. Data were collected from 20 private independent medical practitioners in Mon and Kayin States, Myanmar between December 26, 2010 and January 12, 2011. All areas of assessment showed agreement between OSP and direct observation above 90% except for history taking related to past experience with malaria medicines. In this area, providers did not ask questions of the OSP to the same degree that they questioned real patients (agreement 82.8%).

Conclusions/Significance

The OSP methodology may provide a valuable option for quality assessment of providers in places, or for health conditions, where other assessment tools are unworkable.     

Critical amino acids in syndecan-4 cytoplasmic domain modulation of turkey satellite cell growth and development

Syndecan-4 is composed of a core protein and covalently attached glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein is divided into extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain has two conserved regions and a variable region in the middle. The Ser residue in the conserved region 1 and the Tyr residue in the variable region are important in regulating protein kinase C alpha (PKCα) membrane localization and focal adhesion formation. The objective of the current study was to investigate the role of syndecan-4 Ser and Tyr residues in combination with the GAG and N-glycosylated chains in turkey satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Site-directed mutagenesis was used to generate Ser and Tyr mutants with or without GAG and N-glycosylated chains. The wild type and mutant syndecan-4 constructs were transfected into turkey satellite cells. The over-expression of Ser and Tyr mutants increased cell proliferation and differentiation and decreased membrane localization of PKCα. Furthermore, Ser mutants enhanced cellular responsiveness to FGF2. The results from this study are the first demonstration of a role of syndecan-4 cytoplasmic domain Ser and Tyr residues in regulating satellite cell proliferation, differentiation, and the modulation of cellular responsiveness to FGF2.     

Chloroplast, mitochondrial, and nuclear microsatellites from the southern Appalachian hornwort, Nothoceros aenigmaticus (Dendrocerotaceae)

? Premise of the study: New microsatellite primers were developed for testing genetic differentiation within Nothoceros aenigmaticus and their potential use in other Nothoceros species. The microsatellites are designed to investigate partitioning of genetic variation in a taxon with a peculiar sex allopatry in the southern Appalachian Mountains and relationships with conspecific sexual populations from Mexico. ? Methods and Results: We used two methods for microsatellite development: an enriched library and second-generation shotgun sequence reads. From these two methods, a total of nine primer pairs were selected and tested on 89 southern Appalachian N. aenigmaticus accessions, nine Mexican accessions, and 16 N. vincentianus accessions. Three mitochondrial loci were recovered from the enriched library method and six loci from 454 shotgun sequencing: three were from the chloroplast and three from the nucleus. The primers amplified repeats with two to 20 alleles per locus. ? Conclusions: New microsatellite primers were developed for testing genetic differentiation within N. aenigmaticus and potentially for use in other Nothoceros species. We present one of the first reports of highly polymorphic mitochondrial microsatellites in plants.     

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: State of the science and planning the way forward

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.     

Amino acid uptake in deciduous and coniferous taiga ecosystems

We measured in situ uptake of amino acids and ammonium across deciduous and coniferous taiga forest ecosystems in interior Alaska to examine the idea that late successional (coniferous) forests rely more heavily on dissolved organic nitrogen (DON), than do early successional (deciduous) ecosystems. We traced ¹⁵N-NH₄⁺ and ¹³C-¹⁵N-amino acids from the soil solution into plant roots and soil pools over a 24 h period in stands of early successional willow and late successional black spruce. Late successional soils have much higher concentrations of amino acid in soil solution and a greater ratio of DON to dissolved inorganic N (DIN) (ammonium plus nitrate) than do early successional soils. Moreover, late successional coniferous forests exhibit higher rates of soil proteolytic activity, but lower rates of inorganic N turnover. Differences in ammonium and amino acid uptake by early successional willow stands were insignificant. By contrast, the in situ uptake of amino acid by late successional black spruce forests were approximately 4-fold greater than ammonium uptake. The relative difference in uptake of ammonium and amino acids in these forests was approximately proportional to the relative difference of these N forms in the soil solution. Thus, we suggest that differences in uptake of different N forms across succession in these boreal forests largely reflect edaphic variation in available soil N (composition), rather than any apparent physiological specialization to absorb particular forms of N. These finding are relevant to our understanding of how taiga ecosystems may respond to increases in temperature, fire frequency, N deposition, and other potential consequences of global change.