Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Experimental Comparison of the Clinical Measurement of Ankle Joint Dorsiflexion and Radiographic Tibiotalar Position.

Clinical measurement of ankle dorsiflexion is typically used to diagnose limited ankle range of motion. Controversy and a lack of clarity continue regarding the most accurate clinical method of measuring ankle joint dorsiflexion and the effect that the foot position (supinated, neutral, pronated) has on the true tibiotalar position. We investigated the effects of supinated, neutral and pronated foot positions on the clinical dorsiflexion measurements in 50 healthy subjects and compared these results to the radiographic measurement of tibiotalar joint position with the ankle maximally dorsiflexed in each of the 3 foot positions. Interrater reliability was confirmed to be adequate among the 3 clinicians of varied skill levels. Radiographic measurements of the tibiotalar position showed very little change in each of the 3 foot positions, with a total difference of 0.35° between supination and pronation. However, we found a mean difference of 14° of dorsiflexion in the clinical measurements between the pronated and supinated foot position, with a 9.08° difference between the neutral and supinated positions. Motion of the foot between the neutral and supinated positions introduced an additional source of potential error from the measurement technique when using the neutral position as the standard, which has been recommended in the past. We recommend a supinated foot position as a more reliable foot position for measuring the clinical ankle joint range of motion and propose it as a potential standard.

Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization.

UNLABELLED: New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A'B' and E'F' loops. Six mutations in the E'F' loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCE: Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid: one with a superior fit and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody-virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.

Pyrethroid pesticide exposure and placental effects.

Human exposures to pyrethroid pesticides have increased in recent years following the bans and sanctions placed on other families of pesticides. Although pyrethroids are currently widely used across the United States and throughout the world, and their overt neurological toxicity classified, the extent of their toxicity through low dose and chronic exposures on humans is less well characterized, particularly when it comes to prenatal exposures, their impacts on neurodevelopment, and any role for the placenta in those effects. In this review, we assess the state of research on pyrethroid pesticide exposure and placental effects. These studies presented hormone disrupting, genotoxic, neurodevelopmental and neurobehavioral effects, among others, following prenatal pyrethroid exposures, and highlights a need for future research to assess gaps relating to effects in the human placenta and mechanisms of toxicity as well as shortcomings in the reproducibility and standardization of the methodologies presented.

Management of Subchondral Lesions in the Foot and Ankle.

The treatment of subchondral lesions is an area with limited focus within the foot and ankle literature. The literature has shown an association between disruption of the subchondral bone plate and the formation of subchondral cysts. The primary causes of subchondral lesions are acute trauma, repetitive microtrauma, as well as idiopathic means. Evaluation of these injuries should be done carefully and often requires advanced imaging including MRI and computed tomography. Treatment does vary depending on the presentation of the subchondral lesion with or without the presence of an osteochondral lesion.

Reaction stoichiometry directs the architecture of trimetallic nanostructures produced via galvanic replacement.

Galvanic replacement (GR) of monometallic nanoparticles (NPs) provides a versatile route to interesting bimetallic nanostructures, with examples such as nanoboxes, nanocages, nanoshells, nanorings, and heterodimers reported. The replacement of bimetallic templates by a more noble metal can generate trimetallic nanostructures with different architectures, where the specific structure has been shown to depend on the relative reduction potentials of the participating metals and lattice mismatch between the depositing and template metal phases. Now, the role of reaction stoichiometry is shown to direct the overall architecture of multimetallic nanostructures produced by GR with bimetallic templates. Specifically, the number of initial metal islands deposited on a NP template depends on the reaction stoichiometry. This outcome was established by studying the GR process between intermetallic PdCu (i-PdCu) NPs and either AuCl2- (Au1+) or AuCl4- (Au3+), producing i-PdCu-Au heterostructures. Significantly, multiple Au domains form in the case of GR with AuCl2- while only single Au domains form in the case of AuCl4-. These different NP architectures and their connection to reaction stoichiometry are consistent with Stranski-Krastanov (SK) growth, providing general guidelines on how the conditions of GR processes can be used to achieve multimetallic nanostructures with different defined architectures.

Ankle and Pantalar Arthrodesis: End-Stage Salvage in Cavus Foot.

Bony alignment is the primary goal in foot and ankle reconstruction of the cavovarus foot. This condition presents as a malalignment causing a medial overload of the ankle articular surface and lateral overload of the hindfoot, midfoot, and forefoot. A painful gait associated with articular degeneration of the numerous joints can lead to a chronic and rigid arthrosis of joints, warranting arthrodesis of the affected joints accordingly.

Neurologic Conditions Associated with Cavus Foot Deformity.

The cavus foot deformity is an often less understood deformity within the spectrum of foot and ankle conditions. The hallmark concern is the possibility of an underlying neurologic or neuromuscular disorder. Although a proportion of these deformities are idiopathic, a significant majority do correlate with an underlying disorder. The appropriate evaluation of this deformity, in coordination within the multidisciplinary scope of health care, allows for a timely diagnosis and understanding of the patient's condition. We provide an abbreviated survey of possible underlying etiologies for the patient with the cavus foot deformity as a reference to the foot and ankle surgeon.

Tendon Transfers and Their Role in Cavus Foot Deformity.

Management of the cavus foot is a difficult task for the foot and ankle surgeon. Tendon transfers have been a longstanding accepted treatment for the flexible cavus foot. Performing tendon transfers requires an in-depth understanding of the patient's medical history, factors leading to the development of deformity, as well as the deforming forces contributing to the deformity. Evaluation of the patient for rigid, progressive, and/or spastic deformities is critical to avoid postoperative complications. Educating the patient on postoperative rehabilitation, potential complications, and postoperative expectations is essential to ensure appropriate surgical outcomes.

Phase-Controlled Synthesis of Pd-Sn Nanocrystal Catalysts of Defined Size and Shape.

Bimetallic Pd-based nanoparticles (NPs) are of interest as electrocatalysts for formic acid electrooxidation (FAEO) because of their higher initial catalytic activity and CO tolerance when compared to Pt. Intermetallic NPs (i-NPs) with specific geometric and electronic structures generally exhibit superior catalytic activity, selectivity, and durability when compared to their disordered (random alloy) counterparts; however, the colloidal synthesis of i-NPs remains a challenge. Here, a one-pot method was demonstrated as a facile route to obtain monodisperse Pd-Sn NPs with phase control, including intermetallic hexagonal Pd3Sn2 (P63/mmc), intermetallic orthorhombic Pd2Sn (Pnma), and alloy cubic Pd3Sn (FCC, Fm3m) as size-controlled NPs with quasi-spherical shapes. Initial metal precursor ratios and reaction temperature were critical parameters to achieving phase control. Also, slight modifications of synthetic conditions resulted in either Pd2Sn nanorhombohedra or nanorods with tunable aspect ratios. A systematic evaluation of the Pd-Sn NPs for FAEO showed that most presented higher specific activities when compared to commercial Pd/C, in which Pd2Sn quasi-spheres and nanorhombohedra showed the highest catalytic activity for FAEO. These results highlight the benefits of phase-controlled Pd-based nanocatalysts with defined nanocrystal size and shape, with use of trioctylphospine (TOP) and oleic acid (OA) central to shape and size control.

T-type calcium channel regulation by specific G-protein betagamma subunits.

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.

Perioperative Management of the Rheumatoid Patient.

Rheumatoid arthritis is a complex disease state with multiple associated comorbidities. Perioperative evaluation of the rheumatoid patient from a multidisciplinary approach is necessary to achieve favorable outcomes. A complete history and physical, laboratory, cervical, cardiovascular, pulmonary, and medication assessment before surgery should be performed. Educating the patient on potential complications, such as wound dehiscence, infection, and venous thromboembolism, as well as general postoperative expectations, is essential when evaluating the rheumatoid patient for surgery.

Functional TRPM7 channels accumulate at the plasma membrane in response to fluid flow.

Many cells are constantly exposed to fluid mechanical forces generated by flowing blood, and wall shear stresses modulate aspects of their structure and function. However, the mechanisms for mechanotransduction of flow are not well understood. Here we report that TRPM7, which is both an ion channel and a functional kinase, is translocated within cells in response to laminar flow. After exposure of cells to physiological values of laminar fluid flow, the number of TRPM7 molecules localized at or near the plasma membrane increased up to 2-fold, in less than 100 seconds. This increase in membrane-localized GFP-TRPM7, as seen by total internal reflection fluorescence microscopy, closely correlated with increases in TRPM7 current. Both endogenous and heterologously expressed TRPM7 was found in tubulovesicular structures that were translocated to the region of the plasma membrane on induction of shear stress. In vascular smooth muscle cells, but not in several types of endothelial cells, fluid flow increased endogenous native TRPM7 current amplitude. We hypothesize that TRPM7 plays a role in pathological response to vessel wall injury.

Preservation of protein expression systems at elevated temperatures for portable therapeutic production.

Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill Pseudomonas aeruginosa, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability.

Statistical analysis of co-occurrence patterns in microbial presence-absence datasets.

Drawing on a long history in macroecology, correlation analysis of microbiome datasets is becoming a common practice for identifying relationships or shared ecological niches among bacterial taxa. However, many of the statistical issues that plague such analyses in macroscale communities remain unresolved for microbial communities. Here, we discuss problems in the analysis of microbial species correlations based on presence-absence data. We focus on presence-absence data because this information is more readily obtainable from sequencing studies, especially for whole-genome sequencing, where abundance estimation is still in its infancy. First, we show how Pearson's correlation coefficient (r) and Jaccard's index (J)-two of the most common metrics for correlation analysis of presence-absence data-can contradict each other when applied to a typical microbiome dataset. In our dataset, for example, 14% of species-pairs predicted to be significantly correlated by r were not predicted to be significantly correlated using J, while 37.4% of species-pairs predicted to be significantly correlated by J were not predicted to be significantly correlated using r. Mismatch was particularly common among species-pairs with at least one rare species (<10% prevalence), explaining why r and J might differ more strongly in microbiome datasets, where there are large numbers of rare taxa. Indeed 74% of all species-pairs in our study had at least one rare species. Next, we show how Pearson's correlation coefficient can result in artificial inflation of positive taxon relationships and how this is a particular problem for microbiome studies. We then illustrate how Jaccard's index of similarity (J) can yield improvements over Pearson's correlation coefficient. However, the standard null model for Jaccard's index is flawed, and thus introduces its own set of spurious conclusions. We thus identify a better null model based on a hypergeometric distribution, which appropriately corrects for species prevalence. This model is available from recent statistics literature, and can be used for evaluating the significance of any value of an empirically observed Jaccard's index. The resulting simple, yet effective method for handling correlation analysis of microbial presence-absence datasets provides a robust means of testing and finding relationships and/or shared environmental responses among microbial taxa.

A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II.

Low-voltage-activated (LVA) Ca2+ channels are widely distributed throughout the CNS and are important determinants of neuronal excitability, initiating dendritic and somatic Ca2+ spikes that trigger and shape the pattern of action potential firing. Here, we define a molecular mechanism underlying the dynamic regulation of alpha1H channels (Cav3.2), by Ca2+/CaM-dependent protein kinase II (CaMKII). We show that channel regulation is selective for the LVA alpha1H Ca2+ channel subtype, depends on determinants in the alpha1H II-III intracellular loop, and requires the phosphorylation of a serine residue absent from unregulated alpha1G (Cav3.1) channels. These studies identify the alpha1H channel as a new substrate for CaMKII and provide the first molecular mechanism for the direct regulation of T-type Ca2+ channels by a protein kinase. Our data suggest a novel mechanism for modulating the integrative properties of neurons.

A high-throughput drop microfluidic system for virus culture and analysis.

High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 µm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.

Whole-cell voltage clamp measurements of anthrax toxin pore current.

Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.

Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C).

Molecular cloning of low-voltage activated (LVA) T-type calcium channels has enabled the study of their regulation in heterologous expression systems. Here we investigate the regulation of Ca(v)3.2 alpha(1)-subunits (alpha1H) by calcium- and/or calmodulin-dependent protein kinase II (CaMKII). 293 cells stably expressing alpha1H were transiently transfected with CaMKIIgamma(C). Using the whole-cell recording configuration, we observed that elevation of pipette free Ca(2+) (1 microM) in the presence of CaM (2 microM) increases T-type channel activity selectively at negative potentials, evoking an 11 mV hyperpolarizing shift in the half-maximal potential (V(1/2)) for activation. The V(1/2) of channel inactivation is not altered by Ca(2+)/CaM. These effects reproduced modulation observed in adrenal zona glomerulosa cells. The potentiation by Ca(2+)/CaM was dependent on the co-expression of CaMKIIgamma(C) and required Ca(2+)/CaM-dependent kinase activity. Peptide (AIP) and lipophilic (KN-62) protein kinase inhibitors prevented the Ca(2+)/CaM-induced changes in channel gating without altering basal Ca(v)3.2 channel activity (27 nM free Ca(2+)) as did replacing pipette ATP with adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable analogue. CaMKII-dependent potentiation of channel opening resulted in significant increases in apparent steady-state open probability (P(o)) and sustained channel current at negative voltages. Under identical conditions, CaMKII activation did not regulate the activity of Ca(v)3.1 channels, the first cloned member (alpha1G) of the T-type Ca(2+) channel family. Our results provide the first evidence for the differential regulation of two members of the Ca(v)3 family by protein kinase activation and the first report reconstituting CaMKII-dependent regulation of any cloned Ca(2+) channel.