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It has been a steady year of progress for the New Zealand Medical Students’ Association (NZMSA) in 2019. Last year, our long advocacy campaign with the Government to raise the student loan lifetime limit reached a satisfying conclusion. This result demonstrated the importance of a strong students’ association to carry advocacy across multiple teams and “generations” of medical students. With that issue off our agenda for the first time in eight years, we have had the opportunity to outline some new priorities this year. Alongside our regular advocacy work in the areas of wellbeing, workforce, and education, we have had a strong interest in the role that gender plays in our medical education and the make-up of NZMSA’s leadership. In this review, I will give you a taste of what we have been working on.

Abstract Background The polyphenolic products of the phenylpropanoid pathway, including proanthocyanidins, anthocyanins and flavonols, possess antioxidant properties that may provide health benefits. To investigate the genetic architecture of control of their biosynthesis in apple fruit, various polyphenolic compounds were quantified in progeny from a 'Royal Gala' × 'Braeburn' apple population segregating for antioxidant content, using ultra high performance liquid chromatography of extracts derived from fruit cortex and skin. Results Construction of genetic maps for 'Royal Gala' and 'Braeburn' enabled detection of 79 quantitative trait loci (QTL) for content of 17 fruit polyphenolic compounds. Seven QTL clusters were stable across two years of harvest and included QTLs for content of flavanols, flavonols, anthocyanins and hydroxycinnamic acids. Alignment of the parental genetic maps with the apple whole genome sequence in silico enabled screening for co-segregation with the QTLs of a range of candidate genes coding for enzymes in the polyphenolic biosynthetic pathway. This co-location was confirmed by genetic mapping of markers derived from the gene sequences. Leucoanthocyanidin reductase (LAR1) co-located with a QTL cluster for the fruit flavanols catechin, epicatechin, procyanidin dimer and five unknown procyanidin oligomers identified near the top of linkage group (LG) 16, while hydroxy cinnamate/quinate transferase (HCT/HQT) co-located with a QTL for chlorogenic acid concentration mapping near the bottom of LG 17. Conclusion We conclude that LAR1 and HCT/HQT are likely to influence the concentration of these compounds in apple fruit and provide useful allele-specific markers for marker assisted selection of trees bearing fruit with healthy attributes.

Wolbachia are a genus of insect endosymbiotic bacteria which includes strains w Mel and w AlbB that are being utilized as a biocontrol tool to reduce the incidence of Aedes aegypti -transmitted viral diseases like dengue. However, the precise mechanisms underpinning the antiviral activity of these Wolbachia strains are not well defined. Here, we generated a panel of Ae. aegypti -derived cell lines infected with antiviral strains w Mel and w AlbB or the non-antiviral Wolbachia strain w Pip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while w Pip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in w Mel- and w AlbB-infected cell lines and mosquito tissues compared to cells infected with w Pip or Wolbachia -free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by w Mel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of dengue virus (DENV) replication complexes. Defining Wolbachia 's antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.IMPORTANCE Aedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia , reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid droplets. Inhibiting Wolbachia -induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia's antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells., Competing Interests: The authors declare no conflict of interest.

Background: Introgression of the bacterial endosymbiont Wolbachia into Aedes aegypti populations is a biocontrol approach being used to reduce arbovirus transmission. This requires mass release of Wolbachia-infected mosquitoes. While releases have been conducted using a variety of techniques, egg releases, using water-soluble capsules containing mosquito eggs and larval food, offer an attractive method due to its potential to reduce onsite resource requirements. However, optimisation of this approach is required to ensure there is no detrimental impact on mosquito fitness and to promote successful Wolbachia introgression., Methods: We determined the impact of storage time and temperature on wild-type (WT) and Wolbachia-infected (wMel or wAlbB strains) Ae. aegypti eggs. Eggs were stored inside capsules over 8 weeks at 18 °C or 22 °C and hatch rate, emergence rate and Wolbachia density were determined. We next examined egg quality and Wolbachia density after exposing eggs to 4-40 °C to determine how eggs may be impacted if exposed to extreme temperatures during shipment., Results: Encapsulating eggs for 8 weeks did not negatively impact egg viability or resulting adult emergence and Wolbachia density compared to controls. When eggs were exposed to temperatures within 4-36 °C for 48 h, their viability and resulting adult Wolbachia density were maintained; however, both were significantly reduced when exposed to 40 °C., Conclusions: We describe the time and temperature limits for maintaining viability of Wolbachia-infected Ae. aegypti eggs when encapsulated or exposed to extreme temperatures. These findings could improve the efficiency of mass releases by providing transport and storage constraints to ensure only high-quality material is utilised during field releases., (© 2023. The Author(s).)

Wolbachia is an endosymbiotic bacterium that can restrict the transmission of human pathogenic viruses by Aedes aegypti mosquitoes. Recent field trials have shown that dengue incidence is significantly reduced when Wolbachia is introgressed into the local Ae. aegypti population. Female Ae. aegypti are anautogenous and feed on human blood to produce viable eggs. Herein, we tested whether people who reside on Tri Nguyen Island (TNI), Vietnam developed antibodies to Wolbachia Surface Protein (WSP) following release of Wolbachia -infected Ae. aegypti , as a measure of exposure to Wolbachia . Paired blood samples were collected from 105 participants before and after mosquito releases and anti-WSP titres were measured by ELISA. We determined no change in anti-WSP titres after ~30 weeks of high levels of Wolbachia - Ae. aegypti on TNI. These data suggest that humans are not exposed to the major Wolbachia surface antigen, WSP, following introgression of Wolbachia -infected Ae. aegypti mosquitoes.

Dengue virus (DENV) threatens almost 70% of the world's population, with no effective therapeutic currently available and controversy surrounding the one approved vaccine. A key factor in dengue viral replication is the interaction between DENV nonstructural proteins (NS) 5 and 3 (NS3) in the infected cell. Here, we perform a proof-of-principle high-throughput screen to identify compounds targeting the NS5-NS3 binding interface. We use a range of approaches to show for the first time that two small molecules-repurposed drugs I-OMe tyrphostin AG538 (I-OMe-AG238) and suramin hexasodium (SHS)-inhibit NS5-NS3 binding at low μM concentration through direct binding to NS5 that impacts thermostability. Importantly, both have strong antiviral activity at low μM concentrations against not only DENV-2, but also Zika virus (ZIKV) and West Nile virus (WNV). This work highlights the NS5-NS3 binding interface as a viable target for the development of anti-flaviviral therapeutics.

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia -resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia . While there are hurdles to dengue viruses developing resistance to Wolbachia , we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia -introgression biocontrol programs.

The artificial introduction of the endosymbiotic bacterium, Wolbachia pipientis, into Aedes (Ae.) aegypti mosquitoes reduces the ability of mosquitoes to transmit human pathogenic viruses and is now being developed as a biocontrol tool. Successful introgression of Wolbachia -carrying Ae. aegypti into native mosquito populations at field sites in Australia, Indonesia and Malaysia has been associated with reduced disease prevalence in the treated community. In separate field programs, Wolbachia is also being used as a mosquito population suppression tool, where the release of male only Wolbachia -infected Ae. aegypti prevents the native mosquito population from producing viable eggs, subsequently suppressing the wild population. While these technologies show great promise, they require mass rearing of mosquitoes for implementation on a scale that has not previously been done. In addition, Wolbachia induces some negative fitness effects on Ae. aegypti . While these fitness effects differ depending on the Wolbachia strain present, one of the most consistent and significant impacts is the shortened longevity and viability of eggs. This review examines the body of evidence behind Wolbachia's negative effect on eggs, assesses nutritional parasitism as a key cause and considers how these impacts could be overcome to achieve efficient large-scale rearing of these mosquitoes.

The bacterial endosymbiont Wolbachia is a biocontrol tool that inhibits the ability of the Aedes aegypti mosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that when Wolbachia strains wMel or wAlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand how Wolbachia protects mosquitoes from viral infection. Here, we demonstrate that the Wolbachia strain wPip does not inhibit virus infection in Ae. aegypti. We have leveraged this novel finding, and a panel of Ae. aegypti lines carrying virus-inhibitory (wMel and wAlbB) and non-inhibitory (wPip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism of Wolbachia-mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain's ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define how Wolbachia prevents Ae. aegypti from transmitting viruses., Competing Interests: The authors have declared that no competing interests exist.

Objective : To determine the frequency of physiotherapist-administered aerobic exercise testing/training, the proportion of physiotherapists who administer this testing/training, and the barriers that currently exist across different practice environments. A secondary objective is to identify the learning needs of physiotherapists for the development of an education curriculum in aerobic exercise testing and training with electrocardiograph (ECG) administration and interpretation. Design : National, cross-sectional survey. Participants : Registered physiotherapists practicing in Canada. Results : Out of 137 participants, most (75%) physiotherapists prescribed aerobic exercise on a regular basis (weekly); however, 65% had never conducted an aerobic exercise test. There were no significant differences in frequency of aerobic exercise testing across different practice environments or across years of physiotherapy experience. Physiotherapists perceived the main barriers to aerobic exercise testing as being a lack of equipment/space (78%), time (65%), and knowledge (56%). Although most (82%) were uncomfortable administering 12-lead ECG-monitored aerobic exercise tests, 60% stated they would be interested in learning more about ECG interpretation. Conclusion : This study found that physiotherapists are regularly implementing aerobic exercise. This exercise was infrequently guided by formal aerobic exercise testing, which could increase access to safe and effective exercise within the optimal aerobic training zone. As well, this could facilitate training in patients with cardiovascular diagnoses that require additional testing for medical clearance. Increased ECG training and access to equipment for physiotherapists may augment pre-screening aerobic exercise testing. This training should include learning the key arrhythmias for aerobic exercise test termination as defined by the American College of Sports Medicine.

This paper proposes a novel approach for online, individualized gait analysis, based on an adaptive periodic model of any gait signal. The proposed method learns a model of the gait cycle during online measurement, using a continuous representation that can adapt to inter- and intra-personal variability by creating an individualized model. Once the algorithm has converged to the input signal, key gait events can be identified based on the estimated gait phase and amplitude. The approach is implemented and tested on retirement home resident 6 min walk (6MW) data using wearable accelerometers at the ankle. The proposed approach converges within approximately four gait cycles and achieves 3% error in detecting initial swing events.11 An early version of this work was presented in [1]. A more extensive description of related work and an extended method, including optimization of learning rates, were added to this paper. Further, this paper applies and evaluates the method to a new and much larger gait dataset taken from older adults who each have a variety of medical conditions. Therefore, the experimental protocol was also updated and the results are entirely novel.

Dengue virus (DENV) threatens almost 70% of the world's population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) α/β1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5⁻IMPα/β1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPβ1, but can dissociate preformed IMPα/β1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations against not only DENV-2, but also zika virus and West Nile virus. This work highlights DENV NS5 nuclear targeting as a viable target for anti-flaviviral therapeutics.

Wolbachia pipientis from Drosophila melanogaster (wMel) is an endosymbiotic bacterium that restricts transmission of human pathogenic flaviviruses and alphaviruses, including dengue, Zika, and chikungunya viruses, when introduced into the mosquito vector Aedes aegypti. To date, wMel-infected Ae. aegypti have been released in field trials in 5 countries to evaluate the effectiveness of this strategy for disease control. Despite the success in establishing wMel-infected mosquitoes in wild populations, and the well-characterized antiviral capabilities of wMel, transinfecting different or additional Wolbachia strains into Ae. aegypti may improve disease impact, and perhaps more importantly, could provide a strategy to account for the possible evolution of resistant arboviruses. Here, we report the successful transinfection of Ae. aegypti with the Wolbachia strains wMelCS (D. melanogaster), wRi (D. simulans) and wPip (Culex quinquefasciatus) and assess the effects on Ae. aegypti fitness, cytoplasmic incompatibility, tissue tropism and pathogen blocking in a laboratory setting. The results demonstrate that wMelCS provides a similar degree of protection against dengue virus as wMel following an infectious blood meal, and significantly reduces viral RNA levels beyond that of wMel following a direct challenge with infectious virus in mosquitoes, with no additional fitness cost to the host. The protection provided by wRi is markedly weaker than that of wMelCS, consistent with previous characterisations of these lines in Drosophila, while wPip was found to substantially reduce the fitness of Ae. aegypti. Thus, we determine wMelCS as a key candidate for further testing in field-relevant fitness tests and viremic blood feeding challenges in a clinical setting to determine if it may represent an alternative Wolbachia strain with more desirable attributes than wMel for future field testing.

Anticipatory postural adjustments, executed prior to gait initiation, help preserve lateral stability when stepping. Atypical patterns of anticipatory activity prior to gait initiation may occur in individuals with unilateral impairment (e.g., stroke). This study aimed to determine the prevalence, correlates, and consequences of atypical anticipatory postural adjustment patterns prior to gait initiation in a sub-acute stroke population. Forty independently-ambulatory individuals with sub-acute stroke stood on two force plates and initiated gait at a self-selected speed. Medio-lateral centre of pressure displacement was calculated and used to define anticipatory postural adjustments (shift in medio-lateral centre of pressure >10mm from baseline). Stroke severity, motor recovery, and functional balance and mobility status were also obtained. Three patterns were identified: single (typical), absent (atypical), and multiple (atypical) anticipatory postural adjustments. Thirty-five percent of trials had atypical anticipatory postural adjustments (absent and multiple). Frequency of absent anticipatory postural adjustments was negatively correlated with walking speed. Multiple anticipatory postural adjustments were more prevalent when leading with the non-paretic than the paretic limb. Trials with multiple anticipatory postural adjustments had longer duration of anticipatory postural adjustment and time to foot-off, and shorter unloading time than trials with single anticipatory postural adjustments. A high prevalence of atypical anticipatory control prior to gait initiation was found in individuals with stroke. Temporal differences were identified with multiple anticipatory postural adjustments, indicating altered gait initiation. These findings provide insight into postural control during gait initiation in individuals with sub-acute stroke, and may inform interventions to improve ambulation in this population., (Copyright © 2016 Elsevier B.V. All rights reserved.)

While concurrent augmented visual feedback of the center of pressure (COP) or center of gravity (COG) can improve quiet standing balance control, it is not known whether such feedback improves reactive balance control. Additionally, it is not known whether feedback of the COP or COG is superior. This study aimed to determine whether (1) concurrent augmented feedback can improve reactive balance control, and (2) feedback of the COP or COG is more effective. Forty-eight healthy older adults (60-75 years old) were randomly allocated to one of three groups: feedback of the COP, feedback of the COG, or no feedback. The task was to maintain standing while experiencing 30 s of continuous pseudo-random perturbations delivered by a moving platform. Participants completed 25 trials with or without feedback (acquisition), immediately followed by 5 trials without feedback (immediate transfer); 5 trials without feedback were completed after a 24-h delay (delayed transfer). The root mean square error (RMSE) of COP-COG, electrodermal level, and co-contraction index were compared between the groups and over time. All three groups reduced RMSE and co-contraction index from the start of the acquisition to the transfer tests, and there were no significant between-group differences in RMSE or co-contraction on the transfer tests. Therefore, all three groups learned the task equally well, and improved balance was achieved with practice via a more efficient control strategy. The two feedback groups reduced electrodermal level with practice, but the no-feedback group did not, suggesting that feedback may help to reduce anxiety.

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment., Importance: Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond dynamically to environmental changes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available., Methods: We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition., Results: We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model., Conclusions: 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: To determine whether attending an aerobic fitness program during inpatient stroke rehabilitation is associated with increased participation in physical activity after discharge., Design: This was a prospective cohort study. Patients who received inpatient stroke rehabilitation and were discharged into the community (n = 61; mean age, 65 years) were recruited. Thirty-five participants attended a standardized aerobic fitness program during inpatient rehabilitation, whereas 26 did not. The Physical Activity Scale for Individuals with Physical Disabilities (PASIPD) and adherence to the American College of Sports Medicine (ACSM) guidelines were assessed up to 6 months after discharge., Results: Participants in the fitness group had PASIPD scores and adherence to ACSM guidelines similar to those of participants in the nonfitness group up to 6 months after discharge. There was no significant correlation between volume of exercise performed during the inpatient program and amount of physical activity after discharge., Conclusion: Participation in an inpatient fitness program did not increase participation in physical activity after discharge in individuals with stroke. A new model of care that encourages patients to pursue physical activity after discharge and reduces the potential barriers to participation should be developed.

Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/β (IMPα/β). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMPα/β-recognized nuclear localization signal [Pryor et al. Traffic 8:795-807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8:795-807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMPα/β using an AlphaScreen protein-protein binding assay.

Introduction: It appears that age-related changes in visual attention may impair ability to acquire the visuospatial information needed to grasp a handrail effectively in response to sudden loss of balance. This, in turn, may increase risk of falling. To counter this problem, we developed a proximity-triggered cueing system that provides a visual cue (flashing lights) and/or verbal cue ("attention use the handrail") to attract attention to the handrail. This study examined the effect of handrail cueing on grasping of the rail and associated gaze behavior in a large cohort (n=160) of independent and ambulatory older adults (age 64-80)., Methods: The handrail and cueing system was mounted on a large (2 m×6 m) motion platform configured to simulate a real-life environment. Subjects performed a daily-life task that required walking to the end of the platform, which was triggered to perturb balance by moving suddenly when they were adjacent to the rail. To prevent adaptation, each subject performed only one trial, and a deception was used to ensure that the perturbation was truly unexpected. Each subject was assigned to one of four cue conditions: visual, verbal, multimodal (visual-plus-verbal) or no cue., Results: Verbal cueing attracted overt visual attention to the handrail and markedly increased proactive grasping (prior to the onset of the balance perturbation) particularly when delivered unimodally. Subjects were otherwise much more likely to grasp the rail in reaction to the perturbation. A possible trend for visual cueing to improve the accuracy of these reactions was offset by adverse effects on reaction speed and on frequency of proactive grasping., Conclusions: The results support the viability of using unimodal verbal cueing to reduce fall risk by increasing proactive handrail use. Conversely, they do not strongly support use of visual cueing (either alone or in combination with verbal cueing) and suggest that it may even have adverse effects. Further study is needed to evaluate effects of handrail cueing in a wide range of populations and real-life settings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Infection by one of the 4 distinct serotypes of dengue virus (DENV) threatens >40% of the world's population, with no efficacious vaccine or antiviral agent currently available. DENV replication through the virus-encoded nonstructural protein (NS) 5 protein occurs in the infected cell cytoplasm, but NS5 from DENV2 has thus far been shown to localize strongly in the nucleus throughout infection. Here we use specific antibodies cross-reactive with NS5 from DENV1-4 to demonstrate nuclear localization of NS5 from all DENV serotypes for the first time in both infected as well as transfected cells, although to differing extents. The small-molecule inhibitor Ivermectin was inhibitory towards both DENV 1 and 2 NS5 interaction with its nuclear transporter importin α/β in vitro, and protected against infection from DENV1-4. Ivermectin thus has potential in the clinical setting as a dengue antiviral., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Individuals with stroke fall more often than age-matched controls. Although many focus on the multifactorial nature of falls, the fundamental problem is likely the ability for an individual to generate reactions to recover from a loss of balance. Stepping reactions to recover balance are particularly important to balance recovery, and individuals with stroke have difficulty executing these responses to prevent a fall following a loss of balance., Objective: The purpose of this study is to determine if characteristics of balance recovery steps are related to falls during inpatient stroke rehabilitation., Methods: We conducted a retrospective review of individuals with stroke attending inpatient rehabilitation (n = 136). Details of falls experienced during inpatient rehabilitation were obtained from incident reports, nursing notes, and patient interviews. Stepping reactions were evoked using a "release-from-lean" postural perturbation. Poisson regression was used to determine characteristics of stepping reactions that were related to increased fall frequency relative to length of stay., Results: In all, 20 individuals experienced 29 falls during inpatient rehabilitation. The characteristics of stepping reactions significantly related to increased fall rates were increased frequency of external assistance to prevent a fall to the floor, increased frequency of no-step responses, increased frequency of step responses with inadequate foot clearance, and delayed time to initiate stepping responses., Conclusions: Impaired control of balance recovery steps is related to increased fall rates during inpatient stroke rehabilitation. This study informs the specific features of stepping reactions that can be targeted with physiotherapy intervention during inpatient rehabilitation to improve dynamic stability control and potentially prevent falls.

Computed tomography (CT) plays a pivotal role in the diagnosis of acute stroke and in treatment decision making. CT perfusion imaging performed with intravenous iodinated contrast material allows calculation of the time to peak enhancement, mean transit time, and cerebral blood volume, important parameters for differentiating between an ischemic penumbra, which might benefit from intravascular therapy with thrombolytic agents, and infarcted tissue, which would not benefit from such therapy. Differentiation between the two entities is important because thrombolytic therapy is associated with an increased risk for intracranial hemorrhage. A finding of delay in peak enhancement or increased mean transit time in a region with normal or only slightly abnormal cerebral blood volume is suggestive of an ischemic penumbra; however, accurate interpretation of the CT perfusion parameters may be difficult in the presence of a cerebrovascular anatomic variant or physiologic condition that produces benign oligemia leading to a false appearance of penumbra. For this reason, CT perfusion parameters must be correlated with the clinical history and findings at unenhanced head CT, angiography or CT angiography, and diffusion-weighted magnetic resonance imaging. The authors identify five possible causes of false penumbras, each of which produces a different pattern at imaging: upstream flow restriction, evolution of ischemic change, vascular dysregulation, positioning of the patient's head at an angle during image acquisition, and variant anatomy in the circle of Willis. Familiarity with the imaging patterns and causes of false penumbras may increase the radiologist's confidence in diagnosis and help avoid costly errors in treatment.

Human PSP94 (prostate secretory protein of 94 amino acids) is a major protein synthesized by the prostate gland and secreted in large quantities in seminal fluid. Previous studies have suggested a potential biomedical utility of PSP94 in applications such as diagnosis/prognosis and in treatment of human prostate cancer (PCa). This study was designed to produce a recombinant human PSP94 (rPSP94) to evaluate its clinical and functional role in PCa. We cloned PSP94 cDNA and successfully expressed an active recombinant protein in yeast using Pichia pastoris expression system. A simple purification strategy was established that incorporated combination of membrane ultrafiltration (Pellicon tangential-flow system) and anion exchange chromatography using DE52 resin. The method minimized the technical level of expertise for the production of high quality functional protein. The purified rPSP94 (>98% purity) showed a single band with SDS-PAGE analysis and a peak with a molecular mass (M(r)) of 11,495 kDa using MALDI TOF mass spectrometry (MS). The in vitro competitive binding assays indicated high functional similarity of the rPSP94 with that of its native counterpart. Furthermore, in vivo administration of rPSP94 caused a significant growth inhibition of hormone refractory Mat LyLu tumors in Dunning rat model. Taken together, our data provides evidence for high suitability of the purified rPSP94 for evaluation of its potential diagnostic and therapeutic role in PCa and as a valuable analytical reference standard for clinical studies.

Hyaluronan has great potential in medicine as a biomaterial. However, in its native form, hyaluronan is rapidly metabolized in vivo by free radicals and enzymes such as hyaluronidase, and it is highly soluble. Various methods have been adopted therefore, to modify the physicochemical properties of hyaluronan, while maintaining biocompatibility, and thereby widen its spectrum of therapeutic applications. Hyaluronan has four reactive groups (acetamido, carboxyl, hydroxyl and the reducing end) available for crosslinking to itself or other polymers. Using a variety of crosslinking agents, researchers have developed a host of crosslinked hyaluronan derivatives with an increased in vivo residence time. This chemical modification has enabled the production of gels and films, which can be used in applications such as the prevention of post-surgical adhesions, wound healing and dermal augmentation. We have found that if hyaluronan is crosslinked to itself, or to other polymers (either synthetic or biopolymer), in two stages, then a high degree of crosslinking is achieved, conferring improved biostability. In each of the two stages, the same crosslinking agent is used, but different functional groups are bound by altering the reaction conditions. The novel process can be tailored to yield water insoluble gels and films with a broad range of physical and chemical characteristics, and greater resistance to degradation by hyaluronidase and free radicals. These derivatives are currently undergoing biocompatibility testing, and should ultimately lead to a series of innovative second-generation medical products.

Background: Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets., Methods: Equilibrium binding assay was employed to demonstrate specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell lines. Binding proteins were partially purified by PSP94 affinity-chromatography from LNCaP, PC-3 cells, and prostate tissues., Results: Binding of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competitively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (Kd) and density (Bmax) were determined by Scatchard analysis for each of the two cell lines. For the LNCaP cells, these values were Kd 1 = 0.75 nM and Bmax1 = 300 fmol/mg protein and Kd 2 = 4.5 nM, Bmax2 = 780 fmol/mg protein, respectively. Similar affinity and density results were obtained for PC-3 cells: Kd 1 = 0.83 nM, Bmax1 = 250 fmol/mg protein, and Kd 2 = 5.0 nM, Bmax2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was competitively inhibited by the partially purified proteins. Analysis of these proteins SDS-PAGE showed three main bands and the molecular weights of these three bands were approximately 180, 100 and 60 kD, respectively., Conclusions: The data showed the presence of specific binding proteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue.

A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS-polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18-25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS-PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS-PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.

PSP94 has the potential to be a useful diagnostic marker and therapeutic agent in prostate cancer. Recently, different immunoassay systems for quantitative analysis of PSP94 in clinical samples have been developed, but the epitope structure of PSP94 protein has not been elucidated. In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein. GST-PSP94 contains antigenic determinants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the structure of natural PSP94 antigen. Since GST-PSP94 was expressed in E. coli and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the primary amino acid sequence, rather than conformational structure. This hypothesis was supported by reciprocal competition in ELISA among natural, GST-PSP94 fusion protein, and purified recombinant PSP94 protein. The results demonstrate that the various forms of PSP94 can compete with each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity. When natural and recombinant PSP94 protein were denatured in vitro with urea and alkali, no effect on the binding to antibody was found. The epitope activity of natural PSP94 was also shown to be resistant to the treatment of detergent and reducing agent. The location of one of the linear epitopes recognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequences. Competitive ELISA between the N-terminal peptide and PSP94 protein indicate that both natural and GST-PSP94 have similar immunoactive N-termini.

1. Rainbow trout exposed to seawater or 20 ppm zinc showed hypersecretion of mucus whose sialic acid (N-acetylneuraminic acid) content 0.192 micro M g-1 mucus was not significantly different to that produced by trout in freshwater, 0.184 micro M g-1 mucus. 2. Measurement of sialic content in the water indicated its production (and therefore mucus production) was about 70 times higher in seawater and 20 ppm zinc compared to freshwater production.