Mutagenesis of the dengue virus envelope glycoprotein gene can significantly alter virus infectivity phenotypes in cultured cells and live mosquitoes

2011 Spring.Includes bibliographical references.The dengue virus (DENV) envelope (E) glycoprotein is the primary determinant for initiation of host cell infection. To date, studies investigating the contribution of DENV genetics to mosquito infection are limited. A infectious clone cDNA of DENV type 2 strain 16681 (30P-NBX) provided the ability to introduce site-specific amino acid (AA) mutations into the E protein. The results of the studies herein analyze the effects that AA mutations in the E protein have on infectivity of cultured cells and live mosquitoes. The ability of 30P-NBX to infect Aedes aegypti RexD strain mosquitoes after oral infectious blood-meal was investigated and showed that both 30P-NBX and the parent virus 16681 have low, but equivalent midgut infection rates (MIRs). Mosquito midgut infection with 30P-NBX is not affected by the virus titer in the blood-meal as long as titers are above 6 log 10 pfu/ml or 7 log 10 TCID 50 /ml. Additionally, multiple experimental repetitions with at least 20 mosquitoes per infectious blood-feed were required to obtain an accurate average MIR for 30P-NBX. Serial passage of 30P-NBX in RexD mosquito midguts identified a single AA mutation at position 122 in domain II of the E protein from lysine to glutamic acid that correlated with increased MIRs. Introduction of this AA mutation into the infectious clone (mutant virus K122E) reproduced the results from the serial passage experiment. Compared to 30P-NBX, K122E was not only shown to infect a higher proportion of mosquitoes as early as day 2 post blood-feed, but also to produce a disseminated infection in a higher proportion of mosquitoes by day 6 post blood-feed. Also, K122E consistently produced a midgut infection that spread throughout the entire tissue while 30P-NBX stayed restricted by comparison. Virus attachment to midgut cells was compared and showed that 30P-NBX and K122E could attach with equal efficiencies via our midgut-virus attachment assay. Additionally, incorporation of a single AA mutation into the infectious clone at E protein AA 120 from arginine to threonine significantly enhanced mosquito midgut infection compared to 30P-NBX. This is the first time that mosquito infection determinants have been identified in the DENV E protein. Amino acid mutations were engineered into the E protein on the lateral ridge of domain III, the fusion peptide at the distal end of domain II, and the molecular hinge region between domains I and II. Mutant virus phenotypes were analyzed in cell culture and live mosquitoes. In contrast to previous suggestions, domain III mutant virus phenotypes showed that the FG loop structure (previously suggested as a mosquito-specific infection determinant) and not the specific AA sequence is important for infection of mammalian cells and live mosquitoes, while the structure and sequence of the FG loop is dispensable for infection of cultured C6/36 cells. Additionally, mutations that remove positively charged residues from the A strand in DIII significantly attenuate infection of mosquitoes after oral infectious blood-meal and completely abrogate infection in mammalian cells. The results of this study suggest that there may be multiple structures in the E protein that are contributing to virus-receptor interactions. Viruses with mutations in the fusion peptide and hinge region of the E protein were intrathoracically (IT) inoculated into mosquitoes and showed variable infectivity phenotypes. All of the mutants except for one virus from both the fusion peptide and hinge region viruses attenuated infection of mosquito tissues outside the midgut. Importantly, considering that almost all of these viruses were able to replicate as efficiently as wild type in C6/36 cells, the IT inoculation results provide evidence that C6/36 cells are not a complete surrogate for DENV replication in mosquitoes

A Snapshot of Homelessness and Housing Instability in Michigan Schools

Children need stability to thrive, but for the more than 36,000 children in Michigan's elementary, middle and high schools who face homelessness, stability is often elusive. Under federal education law all children and youth who "lack a fixed, regular, and adequate nighttime residence" are homeless. These children not only lack a stable place to call home, they are more likely to transfer schools, have long commutes, struggle with poor health, and be chronically absent than their non-homeless peers. All of these daily challenges place homeless students at a greater risk for not meeting grade-level standards and for dropping out of school. Recent research in the State of Michigan has shown homelessness among children to be a key factor predicting student achievement in both rural and urban areas, yet little attention has been given, thus far, to understanding where homeless students in Michigan attend school and how their needs might differ depending on their geographic location. This policy brief seeks to fill that gap so that policymakers and local stakeholders can begin to think about the impact of homelessness in their area and to identify resources to support some of the State's most vulnerable children. Data for this brief comes from school year 2015-16 administrative records collected by every school under the mandate of the Federal McKinney-Vento Act, a law which guarantees homeless student's right to an education

Peculiarities of structural-phase state of multi-layer coatings based on Zr-Y-O

Using high-resolution transmission electron microscopy it was established that the Zr-Y-O-based coating layers have a ZrO[2] phase in the tetragonal and monoclinic modifications in the initial state. When it is heated up to 900°C, the ZrO[2] phase in the cubic modification appears in the layer of the coating. With an annealing time of 20 minutes, the grains are crushed to a size of about 15-20 nm. The microdiffraction pattern is modified, the number of reflexes of the tetragonal and monoclinic phases first increases, and then changes without systemic

Structural Loop Between the Cerebellum and the Superior Temporal Sulcus: Evidence from Diffusion Tensor Imaging

The cerebellum is believed to play an essential role in a variety of motor and cognitive functions through reciprocal interaction with the cerebral cortex. Recent findings suggest that cerebellar involvement in the network specialized for visual body motion processing may be mediated through interaction with the right superior temporal sulcus (STS). Yet, the underlying pattern of structural connectivity between the STS and the cerebellum remains unidentified. In the present work, diffusion tensor imaging analysis on seeds derived from functional magnetic resonance imaging during a task on point-light biological motion perception uncovers a structural pathway between the right posterior STS and the left cerebellar lobule Crus I. The findings suggest existence of a structural loop underpinning bidirectional communication between the STS and cerebellum. This connection might also be of potential value for other visual social abilitie

Stressor- and Corticotropin releasing Factor-induced Reinstatement and Active Stress-related Behavioral Responses are Augmented Following Long-access Cocaine Self-administration by Rats

Rationale Stressful events during periods of drug abstinence likely contribute to relapse in cocaine-dependent individuals. Excessive cocaine use may increase susceptibility to stressor-induced relapse through alterations in brain corticotropin-releasing factor (CRF) responsiveness. Objectives This study examined stressor- and CRF-induced cocaine seeking and other stress-related behaviors in rats with different histories of cocaine self-administration (SA). Materials and methods Rats self-administered cocaine under short-access (ShA; 2 h daily) or long-access (LgA; 6 h daily) conditions for 14 days or were provided access to saline and were tested for reinstatement by a stressor (electric footshock), cocaine or an icv injection of CRF and for behavioral responsiveness on the elevated plus maze, in a novel environment and in the light–dark box after a 14- to 17-day extinction/withdrawal period. Results LgA rats showed escalating patterns of cocaine SA and were more susceptible to reinstatement by cocaine, EFS, or icv CRF than ShA rats. Overall, cocaine SA increased activity in the center field of a novel environment, on the open arms of the elevated plus maze, and in the light compartment of a light–dark box. In most cases, the effects of cocaine SA were dependent on the pattern/amount of cocaine intake with statistically significant differences from saline self-administering controls only observed in LgA rats. Conclusions When examined after several weeks of extinction/ withdrawal, cocaine SA promotes a more active pattern of behavior during times of stress that is associated with a heightened susceptibility to stressor-induced cocaine-seeking behavior and may be the consequence of augmented CRF regulation of addiction-related neurocircuitry

Aitchison's Compositional Data Analysis 40 Years On: A Reappraisal

The development of John Aitchison's approach to compositional data analysis is followed since his paper read to the Royal Statistical Society in 1982. Aitchison's logratio approach, which was proposed to solve the problematic aspects of working with data with a fixed sum constraint, is summarized and reappraised. It is maintained that the principles on which this approach was originally built, the main one being subcompositional coherence, are not required to be satisfied exactly -- quasi-coherence is sufficient, that is near enough to being coherent for all practical purposes. This opens up the field to using simpler data transformations, such as power transformations, that permit zero values in the data. The additional principle of exact isometry, which was subsequently introduced and not in Aitchison's original conception, imposed the use of isometric logratio transformations, but these are complicated and problematic to interpret, involving ratios of geometric means. If this principle is regarded as important in certain analytical contexts, for example unsupervised learning, it can be relaxed by showing that regular pairwise logratios, as well as the alternative quasi-coherent transformations, can also be quasi-isometric, meaning they are close enough to exact isometry for all practical purposes. It is concluded that the isometric and related logratio transformations such as pivot logratios are not a prerequisite for good practice, although many authors insist on their obligatory use. This conclusion is fully supported here by case studies in geochemistry and in genomics, where the good performance is demonstrated of pairwise logratios, as originally proposed by Aitchison, or Box-Cox power transforms of the original compositions where no zero replacements are necessary.Comment: 26 pages, 18 figures, plus Supplementary Material. This is a complete revision of the first version of this paper, placing the geochemical example upfront and adding a large section on CoDA of wide matrice

How pressure enhances the critical temperature of superconductivity in YBa2_{2}Cu3_{3}O6+y_{6+y}

High-temperature superconducting cuprates respond to doping with a dome-like dependence of their critical temperature (Tc). But the family-specific maximum Tc can be surpassed by application of pressure, a compelling observation known for decades. We investigate the phenomenon with high-pressure anvil cell NMR and measure the charge content at planar Cu and O, and with it the doping of the ubiquitous CuO2 plane with atomic-scale resolution. We find that pressure increases the overall hole doping, as widely assumed, but when it enhances Tc above what can be achieved by doping, pressure leads to a hole redistribution favoring planar O. This is similar to the observation that the family-specific maximum Tc is higher for materials where the hole content at planar O is higher at the expense of that at planar Cu. The latter reflects dependence of the maximum Tc on the Cu–O bond covalence and the charge-transfer gap. The results presented here indicate that the pressure-induced enhancement of the maximum Tc points to the same mechanism

Safety and efficacy of loteprednol etabonate ophthalmic ointment 0.5% for the treatment of inflammation and pain following cataract surgery

Timothy L Comstock1 Michael R Paterno1 Angele Singh1 Tara Erb1 Elizabeth Davis21Bausch and Lomb Inc., Rochester, NY, USA; 2Minnesota Eye Consultants, Bloomington, MN, USABackground: To compare the safety and efficacy of loteprednol etabonate ophthalmic ointment 0.5% (LE ointment), a new topical ointment formulation, with vehicle for the treatment of inflammation and pain following cataract surgery.Methods: Two randomized, multicenter, double-masked, parallel-group, vehicle-controlled studies were conducted. Patients aged ≥18 years with a combined postoperative anterior chamber cells and flare (ACI) ≥ Grade 3 following uncomplicated cataract surgery participated in seven study visits. Patients self-administered either topical LE ointment or vehicle four times daily for 14 days. Efficacy outcomes included the proportion of patients with complete resolution of ACI and the proportion of patients with no (Grade 0) pain at postoperative day 8. Safety outcomes included the incidence of adverse events, ocular symptoms, changes in intraocular pressure and visual acuity, and biomicroscopy and funduscopy findings.Results: Data from the two studies were combined. The integrated intent-to-treat population consisted of 805 patients (mean [standard deviation] age 69.0 [9.2] years; 58.0% female and 89.7% white). Significantly more LE ointment-treated patients than vehicle-treated patients had complete resolution of ACI (27.7% versus 12.5%) and no pain (75.5% versus 43.1%) at day 8 (P < 0.0001 for both). Fewer LE ointment-treated patients required rescue medication (27.7% versus 63.8%), and fewer had an ocular adverse event (47.2% versus 78.0%, P < 0.0001) while on study treatment. The most common ocular adverse events with LE ointment were anterior chamber inflammation, photophobia, corneal edema, conjunctival hyperemia, eye pain, and iritis. Mean intraocular pressure decreased in both treatment groups. Four patients had increased intraocular pressure ≥10 mmHg (three LE ointment and one vehicle) prior to rescue medication. Visual acuity and dilated funduscopy results were similar between the treatment groups, with the exception of visual acuity at visits 5 and 6, which favored LE ointment.Conclusion: LE ointment was efficacious and well tolerated in the treatment of ocular inflammation and pain following cataract surgery.Keywords: loteprednol etabonate, ophthalmic ointment, postoperative inflammation, postoperative pain, cataract surgery, intraocular pressur