Within-Host Rhinovirus Evolution in Upper and Lower Respiratory Tract Highlights Capsid Variability and Mutation-Independent Compartmentalization.

BACKGROUND: Rhinovirus (RV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how RV evolves within hosts during infection. METHODS: We sequenced RV complete genomes from 12 hematopoietic cell transplant patients with infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL). Metagenomic and amplicon next-generation sequencing were used to track the emergence and evolution of intrahost single nucleotide variants (iSNVs). RESULTS: Identical RV intrahost populations in matched NW and BAL specimens indicated no genetic adaptation is required for RV to progress from URT to LRT. Coding iSNVs were 2.3-fold more prevalent in capsid over nonstructural genes. iSNVs modeled were significantly more likely to be found in capsid surface residues, but were not preferentially located in known RV-neutralizing antibody epitopes. Newly emergent, genotype-matched iSNV haplotypes from immunocompromised individuals in 2008-2010 could be detected in Seattle-area community RV sequences in 2020-2021. CONCLUSIONS: RV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, RV sequences.

Within-host rhinovirus evolution in upper and lower respiratory tract highlights capsid variability and mutation-independent compartmentalization.

Background: Human rhinovirus (HRV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how HRV evolves within hosts during infection. Methods: We sequenced HRV complete genomes from 12 hematopoietic cell transplant patients with prolonged infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL) specimens. Metagenomic (mNGS) and amplicon-based NGS were used to study the emergence and evolution of intra-host single nucleotide variants (iSNVs). Results: Identical HRV intra-host populations in matched NW and BAL specimens indicated no genetic adaptation is required for HRV to progress from URT to LRT. Microbial composition between matched NW and BAL confirmed no cross-contamination during sampling procedure. Coding iSNVs were 2.3-fold more prevalent in capsid over non-structural genes, adjusted for length. iSNVs modeled onto HRV capsid structures were significantly more likely to be found in surface residues, but were not preferentially located in known HRV neutralizing antibody epitopes. Newly emergent, serotype-matched iSNV haplotypes from immunocompromised individuals from 2008-2010 could be detected in Seattle-area community HRV sequences from 2020-2021. Conclusion: HRV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, HRV sequences.

Determining mushroom tyrosinase inhibition by imidazolium ionic liquids: A spectroscopic and molecular docking study.

The inhibition effects of imidazolium ionic liquids (ILs) on the enzyme kinetics of mushroom tyrosinase is reported. A simple UV-VIS spectrophotometric assay was used to measure the reaction kinetics of the reaction between mushroom tyrosinase and L-dopa. Seven different imidazolium ILs, comprised of 1-alkyl-3-methylimidazolium ([Imn1+], n=2, 4, 6) cations paired with several anions that included Cl-, [NO3-], methanesulfonate ([MeSO3-]), trifluoromethanesulfonate (or triflate, [TFMS-]), and bis(trifluoromethylsulfonyl)imide ([Tf2N-]). Lineweaver-Burk plots were generated from the recovered kcat and Km parameters using four to six substrate concentrations per measurement. The results show that mushroom tyrosinase activity was consistently inhibited by all of the ILs and that the type of inhibition was non-competitive in nearly all cases. Only the data for [Im21+][Tf2N-] suggested that the inhibition mechanism was competitive with the substrate. Molecular docking simulations were performed using AutoDock4.2 and AutoDock Vina and revealed that all cations docked in the L-dopa active site. Anions showed varied results that included locations both within and outside of the active site.

Retaining clinician-scientists: nature versus nurture.

In their IOVS article "Rejuvenating Clinician-Scientist Training" (published March 28, 2014), Balamurali Ambati and Judd Cahoon rightly point out the dearth of new clinician-scientists in ophthalmology. Within the context of their suggestions for increasing the number of successful clinician-scientists, they claim that the traditional MD-PhD training programs and K awards have failed to produce individuals who will carry on the important work of clinically relevant research that will improve patients' lives and sight. In this response we present data, including information on the career paths of graduates of the Washington University ophthalmology residency, that call into question the presumed failure of MD-PhD and K award programs and show that, in fact, graduates of these programs are more likely to succeed as clinician-scientists than are their peers who have not trained in such scientifically rigorous environments. We propose that, rather than a failure of early training programs, it may be obstacles that arise later in training and among junior faculty that prevent promising careers from reaching maturity. Funding, one rather large obstacle, takes the form of imbalanced start-up monies, less National Institutes of Health (NIH) funding awarded to young investigators, and study section composition that may work against those with clinically driven questions. We also explore the challenges faced in the culture surrounding residency and fellowship training. We agree with Ambati and Cahoon that there needs to be more innovation in the way training programs are structured, but we believe that the evidence supports supplementing the current model rather than scrapping it and starting over with unproven initiatives. The data on training programs supports the contention that those who have already made substantial investment and commitment to the clinician-scientist pathway through participation in MSTP or K training programs are the most likely to succeed on this career trajectory. To muffle the siren song of private practice and retain those best prepared for the clinician-scientist pathway requires additional investment as their careers mature through protected research time, mentorship, and advocacy.

Comparison of vertical and horizontal saccade measures and their relation to gray matter changes in premanifest and manifest Huntington disease.

Saccades are a potentially important biomarker of Huntington disease (HD) progression, as saccadic abnormalities can be detected both cross-sectionally and longitudinally. Although vertical saccadic impairment was reported decades ago, recent studies have focused on horizontal saccades. This study investigated antisaccade (AS) and memory guided saccade (MG) impairment in both the horizontal and vertical directions in individuals with the disease-causing CAG expansion (CAG+; n = 74), using those without the expansion (CAG-; n = 47) as controls. Percentage of errors, latency, and variability of latency were used to measure saccadic performance. We evaluated the benefits of measuring saccades in both directions by comparing effect sizes of horizontal and vertical measures, and by investigating the correlation of saccadic measures with underlying gray matter loss. Consistent with previous studies, AS and MG impairments were detected prior to the onset of manifest disease. Furthermore, the largest effect sizes were found for vertical saccades. A subset of participants (12 CAG-, 12 premanifest CAG+, 7 manifest HD) underwent magnetic resonance imaging, and an automated parcellation and segmentation procedure was used to extract thickness and volume measures in saccade-generating and inhibiting regions. These measures were then tested for associations with saccadic impairment. Latency of vertical AS was significantly associated with atrophy in the left superior frontal gyrus, left inferior parietal lobule, and bilateral caudate nuclei. This study suggests an important role for measuring vertical saccades. Vertical saccades may possess more statistical power than horizontal saccades, and the latency of vertical AS is associated with gray matter loss in both cortical and subcortical regions important in saccade function.

Abnormal error-related antisaccade activation in premanifest and early manifest Huntington disease.

OBJECTIVE: Individuals with the trinucleotide CAG expansion (CAG+) that causes Huntington's disease (HD) have impaired performance on antisaccade (AS) tasks that require directing gaze in the mirror opposite direction of visual targets. This study aimed to identify the neural substrates underlying altered antisaccadic performance. METHOD: Three groups of participants were recruited: (1) Imminent and early manifest HD (early HD, n = 8); (2) premanifest (presymptomatic) CAG+ (preHD, n = 10); and (3) CAG unexpanded (CAG-) controls (n = 12). All participants completed a uniform study visit that included a neurological evaluation, neuropsychological battery, molecular testing, and functional MRI during an AS task. The blood oxygenation level dependent (BOLD) response was obtained during saccade preparation and saccade execution for both correct and incorrect responses using regression analysis. RESULTS: Significant group differences in BOLD response were observed when comparing incorrect AS to correct AS execution. Specifically, as the percentage of incorrect AS increased, BOLD responses in the CAG- group decreased progressively in a well-documented reward detection network that includes the presupplementary motor area and dorsal anterior cingulate cortex. In contrast, AS errors in the preHD and early HD groups lacked this relationship with BOLD signal in the error detection network, and BOLD responses to AS errors were smaller in the two CAG+ groups as compared with the CAG- group. CONCLUSIONS: These results are the first to suggest that abnormalities in an error-related response network may underlie early changes in AS eye movements in premanifest and early manifest HD. (PsycINFO Database Record (c) 2011 APA, all rights reserved).

Progression in prediagnostic Huntington disease.

OBJECTIVE: To examine rates of decline in individuals at risk for Huntington disease (HD). METHODS: 106 individuals at risk for HD completed a battery of neurocognitive, psychomotor and oculomotor tasks at two visits, approximately 2.5 years apart. Participants were classified as: (1) without the CAG expansion (normal controls, NC; n=68) or (2) with the CAG expansion (CAG+; n=38). The CAG+ group was further subdivided into those near to (near; n=19) or far from (far; n=19) their estimated age of onset. Longitudinal performance in the CAG+ group was evaluated with a repeated measures model with two main effects (time to onset, visit) and their interaction. Analysis of covariance was employed to detect differences in longitudinal performance in the three groups (NC, near and far). RESULTS: In the CAG+, the interaction term was significant (p < or = 0.02) for four measures (movement time, alternate button tapping, variability of latency for a memory guided task and percentage of errors for a more complex memory guided task), suggesting the rate of decline was more rapid as subjects approached onset. Longitudinal progression in the three groups differed for several variables (p<0.05). In most, the near group had significantly faster progression than NC; however, comparisons of the NC and far groups were less consistent. CONCLUSIONS: Different patterns of progression were observed during the prediagnostic period. For some measures, CAG+ subjects closer to estimated onset showed a more rapid decline while for other measures the CAG+ group had a constant rate of decline throughout the prediagnostic period that was more rapid than in NC.

Multiple step pattern as a biomarker in Parkinson disease.

OBJECTIVE: To evaluate quantitative measures of saccades as possible biomarkers in early stages of Parkinson disease (PD) and in a population at-risk for PD. METHODS: The study sample (n=68) included mildly to moderately affected PD patients, their unaffected siblings, and control individuals. All participants completed a clinical evaluation by a movement disorder neurologist. Genotyping of the G2019S mutation in the LRRK2 gene was performed in the PD patients and their unaffected siblings. A high resolution, video-based eye tracking system was employed to record eye positions during a battery of visually guided, anti-saccadic (AS), and two memory-guided (MG) tasks. Saccade measures (latency, velocity, gain, error rate, and multiple step pattern) were quantified. RESULTS: PD patients and a subgroup of their unaffected siblings had an abnormally high incidence of multiple step patterns (MSP) and reduced gain of saccades as compared with controls. The abnormalities were most pronounced in the more challenging version of the MG task. For this task, the MSP measure demonstrated good sensitivity (87%) and excellent specificity (96%) in the ability to discriminate PD patients from controls. PD patients and their siblings also made more errors in the AS task. CONCLUSIONS: Abnormalities in eye movement measures appear to be sensitive and specific measures in PD patients as well as a subset of those at-risk for PD. The inclusion of quantitative laboratory testing of saccadic movements may increase the sensitivity of the neurological examination to identify individuals who are at greater risk for PD.

Cooperative interactions between R531 and acidic residues in the voltage sensing module of hERG1 channels.

HERG1 K(+) channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K(+) channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 - S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.

The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel.

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K(+) current I(K1) and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I(K1). The voltage- and K(+)-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I(K1) even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.

The S4-S5 linker directly couples voltage sensor movement to the activation gate in the human ether-a'-go-go-related gene (hERG) K+ channel.

A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K+ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.

A comparison of Thermo Electron RSV OIA to viral culture and direct fluorescent assay testing for respiratory syncytial virus.

BACKGROUND: Rapid diagnostic methods for respiratory syncytial virus are useful tools available for the clinician. OBJECTIVES: The Thermo Electron RSV OIA (optical immunoassay kit) was prospectively compared with direct immunofluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah. STUDY DESIGN: Specimens from three hundred and thirty patients exhibiting respiratory symptoms were collected for testing by the three methods above. Several specimens were positive by both OIA and DFA with a negative culture result. These culture results were verified by RT-PCR analysis. RESULTS: Overall, 107 specimens were positive for RSV by the reference tests (culture or RT-PCR). DFA analysis identified an additional 40 patient specimens positive for other respiratory viruses. Compared to the reference tests the sensitivity, specificity, positive, and negative predictive values of the OIA for detection of RSV were 87.9%, 99.6%, 98.9% and 94.5%, respectively. CONCLUSIONS: The rapid OIA assay format proved to be cost effective, and simple to use in comparison to DFA and viral culture. Negative rapid test results should still be confirmed with a secondary test.