Non-Bulk-Like Solvent Behavior in the Ribosome Exit Tunnel

As nascent proteins are synthesized by the ribosome, they depart via an exit tunnel running through the center of the large subunit. The exit tunnel likely plays an important part in various aspects of translation. Although water plays a key role in many bio-molecular processes, the nature of water confined to the exit tunnel has remained unknown. Furthermore, solvent in biological cavities has traditionally been characterized as either a continuous dielectric fluid, or a discrete tightly bound molecule. Using atomistic molecular dynamics simulations, we predict that the thermodynamic and kinetic properties of water confined within the ribosome exit tunnel are quite different from this simple two-state model. We find that the tunnel creates a complex microenvironment for the solvent resulting in perturbed rotational dynamics and heterogenous dielectric behavior. This gives rise to a very rugged solvation landscape and significantly retarded solvent diffusion. We discuss how this non-bulk-like solvent is likely to affect important biophysical processes such as sequence dependent stalling, co-translational folding, and antibiotic binding. We conclude with a discussion of the general applicability of these results to other biological cavities

Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm (“codon harmonization”) for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the “recoded” genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli

A Bedside Clinical Prediction Rule for Detecting Moderate or Severe Aortic Stenosis

OBJECTIVE: To evaluate a bedside clinical prediction rule for detecting moderate or severe aortic stenosis. DESIGN: Cross-sectional study with independent comparison to a diagnostic reference standard, doppler echocardiography. SETTING: Urban university hospital. PARTICIPANTS: Consecutive hospital inpatients (n = 124) who had been referred for echocardiography. MEASUREMENTS AND MAIN RESULTS: Participants were examined by a third-year general internal medicine resident and a staff general internist. We hypothesized in advance that absence of a murmur over the right clavicle would rule out aortic stenosis, while the presence of three or four associated findings (slow carotid artery upstroke, reduced carotid artery volume, maximal murmur intensity at the second right intercostal space, and reduced intensity of the second heart sound) would rule in aortic stenosis. Study physicians were unaware of echocardiographic findings. The outcome was echocardiographic moderate or severe aortic stenosis, defined as a valve area of 1.2 cm(2)or less, or a peak instantaneous gradient of 25 mm Hg or greater. Absence of a murmur over the right clavicle ruled out aortic stenosis (likelihood ratio [LR] 0.10; 95% confidence interval [CI] 0.01, 0.44). The presence of three or four associated findings ruled in aortic stenosis (LR 40; 95% CI 6.6, 240). If a murmur was present over the right clavicle, but no more than two associated findings were present, then the examination was indeterminate (LR 1.8; 95% CI 0.93, 2.9). CONCLUSION: A clinical prediction rule, using simple bedside maneuvers, accurately ruled in and ruled out aortic stenosis

Assessment of Patient Capacity to Consent to Treatment

OBJECTIVE: To compare results of a specific capacity assessment administered by the treating clinician, and a Standardized Mini-Mental Status Examination (SMMSE), with the results of expert assessments of patient capacity to consent to treatment. DESIGN: Cross-sectional study with independent comparison to expert capacity assessments. SETTING: Inpatient medical wards at an academic secondary and tertiary referral hospital. PARTICIPANTS: One hundred consecutive inpatients facing a decision about a major medical treatment or an invasive medical procedure. Participants either were refusing treatment, or were accepting treatment but were not clearly capable according to the treating clinician. MEASUREMENTS AND MAIN RESULTS: The treating clinician (medical resident or student) conducted a specific capacity assessment on each participant, using a decisional aid called the Aid to Capacity Evaluation. A specific capacity assessment is a semistructured evaluation of the participant’s ability to understand relevant information and appreciate reasonably foreseeable consequences with regard to the specific treatment decision. Participants also received a SMMSE administered by a research nurse. Participants then had two independent expert assessments of capacity. If the two expert assessments disagreed, then an independent adjudication panel resolved the disagreement after reviewing videotapes of both expert assessments. Using the two expert assessments and the adjudication panel as the reference standard, we calculated areas under the receiver-operating characteristic curves and likelihood ratios. The areas under the receiver-operating characteristic curves were 0.90 for specific capacity assessment by treating clinician and 0.93 for SMMSE score (2p = .48). For the treating clinician’s specific capacity assessment, likelihood ratios for detecting incapacity were as follows: definitely incapable, 20 (95% confidence interval [CI] 3.6, 120); probably incapable, 6.1 (95% CI 2.6, 15); probably capable, 0.39 (95% CI 0.18, 0.81); and definitely capable, 0.05 (95% CI 0.01, 0.29). For the SMMSE, a score of 0 to 16 had a likelihood ratio of 15 (95% CI 5.3, 44), a score of 17 to 23 had a likelihood ratio of 0.68 (95% CI 0.35, 1.2), and a score of 24 to 30 had a likelihood ratio of 0.05 (95% CI 0.01, 0.26). CONCLUSIONS: Specific capacity assessments by the treating clinician and SMMSE scores agree closely with results of expert assessments of capacity. Clinicians can use these practical, flexible, and evaluated measures as the initial step in the assessment of patient capacity to consent to treatment

Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (~1900 years ago), and to the Neolithic era for bioprocesses (~4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses