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Like [alpha][beta] T cells, human [gamma][delta] T cells also have different subsets with distinct characteristics. Whether human V[gamma]9V[delta]2 T cells have functionally different subsets in response to influenza A (fluA) viruses remains unknown. In this study, we show for the first time that both central (CD45RA(-)CD27(+)) and effector (CD45RA(-)CD27(-)) memory V[gamma]9V[delta]2 T cells have similar levels of immediate interferon (IFN) [gamma] and cytotoxic responses to human and avian fluA virus-infected cells. In contrast, CD56(+) V[gamma]9V[delta]2 T cells have significantly higher cytotoxicity against fluA virus-infected cells compared with their CD56(-) counterparts, whereas both subsets have similar IFN-[gamma] responses. We further demonstrate that the CD16-dependent degranulation pathway, but not antibody-dependent cell-mediated cytotoxicity, contribute to the superior cytotoxicity of CD56(+) V[gamma]9V[delta]2 T cells. Our study provides further evidence for the phenotypic and functional characterization of human V[gamma]9V[delta]2 T-cell subsets during fluA virus infection and may help improve the [gamma][delta] T-cell-based immunotherapy for viral infection. [ABSTRACT FROM AUTHOR]

Severe human disease associated with influenza A H5N1 virus was first detected in Hong Kong in 1997. Its recent reemergence in Asia and high associated mortality highlight the need to understand its pathogenesis. We investigated the roles of death receptor ligands (DRLs) in H5N1 infection. Significant up-regulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-alpha, but not Fas ligand (FasL) mRNA, was detected in human monocyte-derived macrophages (MDMs) infected with avian influenza viruses A/Hong Kong/483/97 (H5N1/97) or its precursor, A/Quail/Hong Kong/G1/97. H5N1/97-infected MDMs exhibited the strongest induction of apoptosis in Jurkat T cells, and it could be reduced by TRAIL-receptor 2 blocking antibody. Furthermore, influenza virus infection enhanced the sensitivity of Jurkat T cells to apoptosis induced by TNF-alpha, TRAIL, and FasL. Our data suggested that functional TRAIL produced by influenza virus-infected MDMs was related to their cytotoxicity and that the enhanced sensitization to DRL-induced apoptosis detected in avian influenza may contribute to disease pathogenesis. [ABSTRACT FROM AUTHOR]

Distortion product otoacoustic emissions (DPOAEs) are regularly used to measure cochlear status in the high frequency range, as when monitoring ototoxic medication or for assessing noise-induced hearing loss. This study measured default DP-gram protocols and user-defined DP spectrum protocols in 35 normal hearing young adults using Otodynamics ILO96 equipment. Very short-term and short-term reliability was assessed, with measures conducted 20 minutes, and an average of 15 days later, respectively. The test resolution setting for the DP-gram measurements was varied between 1 to 8 points per octave in each test series. DPOAE amplitudes in the default frequency range (1 to 6 kHz) between test and short-term retest were highly correlated, with average r=0.81. Standard error of measurement (SEM) was found to average 2.64 dB for DPOAE amplitude in the default frequency range. DPOAE amplitudes in the higher frequency range of 6.5 to 7 kHz were also significantly correlated between test and short-term retest; r ranged from 0.80 to 0.82 and SEM ranged from 2.59 to 3.04 dB. The various resolution and retest conditions showed no significant differences in reliability. DPOAE measurement in the 6.5 to 7 kHz range can be considered a reliable tool for monitoring cochlear function in cases such as exposure to ototoxic medication or noise. [ABSTRACT FROM AUTHOR]

Purpose: Our study used preoperative neuroanatomical features to predict auditory development in Chinese-learning children with cochlear implants (CIs)., Method: T1-weighted whole-brain magnetic resonance imaging (MRI) scans were obtained from 17 Chinese-learning pediatric CI candidates (12 females and five males, age at MRI = 23.0 ± 15.0 months). Voxel-based morphometry was applied to examine the children's whole-brain structure. Machine learning was employed using neuroanatomical features to predict children's auditory skills up to 24 months after CI. The whole-brain neural model and auditory/visual cortex neural model were compared with a nonneural model using gender, age at CI activation, and preoperative residual hearing as predictors. Model performance was quantified using the mean square error ( MSE ) between predicted values and observations., Results: The model with preoperative neuroanatomical features showed a significantly smaller MSE than the nonneural model in predicting auditory skills in children with CIs. Specifically, the auditory-related area played an important role in predicting post-CI outcomes., Conclusions: The preoperative neuroanatomical features outperformed the nonneural features in predicting auditory skills in children with CIs. These results indicate that neural structure holds the potential to serve as an objective and effective feature for predicting post-CI outcomes., Supplemental Material: https://doi.org/10.23641/asha.28012046.

Purpose: To investigate 2-year post-operative hearing performance, safety, and patient-reported outcomes of hearing-impaired adults treated with the Osia ® 2 System, an active osseointegrated bone-conduction hearing implant that uses piezoelectric technology., Methods: A prospective, multicenter, open-label, single-arm, within-subject clinical study conducted at three tertiary referral clinical centers located in Melbourne, Sydney and Hong Kong. Twenty adult recipients of the Osia 2 System were enrolled and followed up between 12 and 24 months post-implantation: 17 with mixed or conductive hearing loss and 3 with single-sided sensorineural deafness. Safety data, audiological thresholds, speech recognition thresholds in noise, and patient-reported outcomes were collected and evaluated. In addition, pre-and 6-month post-implantation data were collected retrospectively for this recipient cohort enrolled into the earlier study (ClinicalTrials.gov NCT04041700)., Results: Between 6- and 24-month follow-up, there was no statistically significant change in free-field hearing thresholds or speech reception thresholds in noise (p =  > 0.05), indicating that aided improvements were maintained up to 24 months of follow-up. Furthermore, improvements in health-related quality of life and daily hearing ability, as well as clinical and subjective measures of hearing benefit remained stable over the 24-month period. No serious adverse events were reported during extended follow-up., Conclusions: These study results provide further evidence to support the longer term clinical safety, hearing performance, and patient-related benefits of the Osia 2 System in patients with either a conductive hearing loss, mixed hearing loss, or single-sided sensorineural deafness., Trial Registration: ClinicalTrials.gov Identifier: NCT04754477. First posted: February 15, 2021., (© 2023. The Author(s).)

Introduction: This study aimed at exploring the feasibility of applying a computer-based language test to young children aged 2-4 years., Methods: Thirty-two Cantonese-speaking children, aged 2-4 years, were recruited from local kindergartens. All participants underwent an assessment using both the computer-based and paper-pencil versions of the Macau Cantonese Language Screening Scale for Preschool Children, following a crossover study design. A short break of 15-30 min was provided between the two assessments. The data were analysed at three levels: the overall test, subcategory, and individual item levels. At the overall test and subcategory levels, data were analysed using the paired samples t-test and ICC. At the item level, the percentage of agreement and Cohen's kappa value were selected to assess the agreement of the two test formats., Results: Excellent agreement was found for the overall test level, and good agreement was observed for four of the five subcategories. At the individual item level, 28 of the 35 items showed more than 80% agreement, and 16 items showed substantial to almost perfect agreement., Conclusion: These results suggest that the two test formats give similar total scores and subcategory scores for children aged 2-4. For children older than 2 years and 6 months, the agreement for matching items is as high as 83.68% (1,318/1,575). The computer-based test is thus highly recommended for this group of children. For children younger than 2 years and 6 months, a modified computer-based test is suggested to accommodate their needs., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Purpose: This study investigated to what extent residual hearing and rehabilitation options (e.g., hearing aids [HAs]) affect the auditory cortex in children with hearing loss., Method: Twenty-one children with bilateral congenital sensorineural hearing loss who were candidates for cochlear implantation were recruited. Voxel-based morphometry analysis was conducted to assess the gray matter (GM) volume in the auditory cortex. Children's residual hearing was measured by pure-tone audiometry at different frequencies. Multiple linear regression models were conducted to examine the effects of residual hearing and the use of HAs on GM volume in the auditory cortex with the control of age and gender., Results: Children with more residual hearing at high frequencies had larger GM volume ratio (corrected by total intracranial volume) in the left Heschl's gyrus ( r = -.545, p = .013). An interaction effect between residual hearing and the use of HAs suggested that the effect of residual hearing on GM ratio was moderated by the use of HAs (β = -.791, p = .020). Compared with children with less residual hearing, children who had more residual hearing benefited more from longer use of HAs in terms of a larger GM ratio., Conclusions: Our preliminary findings highlight the impact of residual hearing on the neuroanatomy of the auditory cortex in children with hearing loss. Moreover, our results call for more auditory input via HAs for children with more residual hearing to preserve the auditory cortex before cochlear implantation. For children with less residual hearing who might receive limited benefit from HAs, an early cochlear implant would be necessary.

This study examined the early pragmatic language skills in typically developing (TD) preschool-age children, children with language impairment (LI) and children with autism spectrum disorder (ASD). Two hundred and sixty-two TD children, 73 children with LI, and 16 children with ASD were compared on early pragmatics through direct assessment (DA). Post hoc analysis revealed that children in two clinical groups displayed significant pragmatic language deficits. Children in the ASD group who were older exhibited comparable degree of impairments as their LI peers, suggesting a relatively stagnant development of pragmatic language skills in children with ASD. Findings also supported the use of DA in identifying pragmatic language deficits, which have implications for the adoption of this assessment approach in clinical settings., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Tinnitus is a common problem worldwide. There is still no effective method to cure it. Traditional Chinese medicine (TCM) may be a potentially effective treatment approach for tinnitus. However, there is still no clinical trial with scientifically rigorous methodology to evaluate the treatment effect of TCM for tinnitus. Therefore, we propose a pilot study to inform the feasibility of a future full-scale RCT to establish the efficacy of TCM formula for tinnitus. Objectives: The aim of this study is to determine the feasibility of a full-scale RCT and explore whether a TCM formula (BHT) has an additional effect on improving tinnitus when compared to informative counseling alone. Design: An assessor-blinded, randomized, controlled clinical trial is used. Participants: Twenty-four patients with chronic subjective tinnitus will be enrolled. Interventions: The patients will be allocated randomly to receive a TCM formula (BHT, Bushen Huoxue Tongluo) and informative counseling or informative counseling alone. The oral BHT herbal granules will be taken twice per day continuously for 8 weeks. Main outcome measures: The primary outcomes include recruitment rate, intervention completion rate, and data completion rate to evaluate the feasibility. The secondary outcomes include Tinnitus Handicap Inventory, tinnitus functional index, tinnitus sensation level, self-rated visual analogue scale on tinnitus loudness and annoyance, Pittsburgh Sleep Quality Index, Hospital Anxiety and Depression Scale, and adverse event. The outcome measures will be collected at baseline, end of treatment, and 4-week follow-up. Discussion : This trial is currently ongoing and is recruiting patients. The expected study results will find some preliminary evidence about the clinical effectiveness of BHT on chronic tinnitus and will also determine if it is feasible to conduct a full-scale RCT of BHT and identify the necessary changes to the protocol if possible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Yeung, Tong, Lin, Chang, Ng, Sum, Leung, Chan, Ngan and Tong.)

Background: Life Goals Programme (LGP) was developed as a psychological intervention for bipolar disorder, with its structured 6-session psychoeducation phase (Phase 1) targeting understanding of the disorder, medication adherence, early warning signs, and coping with symptoms and triggers. The present study tested the efficacy of Phase 1 of the LGP on symptom recovery and moment-by-moment mood stability, as well as medication adherence and quality of life., Methods: Adults with bipolar disorder were randomly allocated to the LGP condition (six weekly group sessions) or the waitlist condition (six weeks of standard care, followed by the same LGP intervention). Participants were assessed before and after treatment, and at 6-month follow up. Waitlist participants were additionally assessed at baseline. Assessment included a clinical interview (SCID, MADRS, YMRS, and HAM-A), self-reported questionnaires, and 6-day experience sampling assessment on a mobile device., Results: 64 out-patients with bipolar disorder (44 Bipolar I and 20 Bipolar II subtypes) participated in this study. LGP was well received and attended. Following LGP, there were significant improvements in knowledge about illness and level of anxiety, which were of large effect sizes and sustained at 6-month follow-up. Experience sampling assessment revealed small but significant improvements in moment-by-moment mood stability. Improvement in medication adherence was significant at 6 months and was of a moderate-to-large effect size. Changes in anxiety and mood stability were significantly greater following LGP than standard care., Limitations: Our results warrant further testing against active control., Conclusions: There was a robust improvement in emotional regulation following the 6-week LGP., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Despite evidence that excessive screen use may contribute to negative health, developmental, emotional, and behavioral outcomes, more children are engaging in increasing amounts of screen-related activities. For children with neurodevelopmental conditions, increased screen use could exacerbate emotional/behavioral difficulties (EBDs) by interfering with sleep quantity and quality., Aims: This study examined the possible mediating role of sleep in the relationship between screen use and EBDs in preschool children with neurodevelopmental disorders (NDDs) clinically referred to a child development center in Singapore., Methods: A screen use questionnaire developed for the purposes of the present study, the Children's Sleep Habits Questionnaire, and the Strengths and Difficulties Questionnaire were completed by 367 caregivers of 2- to 5-year-old children with NDDs (39.5% autism spectrum disorder; 36.8% speech-language disorders; 23.7% others)., Results: Average daily screen use duration was 3.98 hours, with 93.9% exceeding 1 hour of screen time daily. 57.7% of children had screen devices in their bedrooms, while 52% commenced screen use at the age of 18 months or earlier. Sleep problems fully mediated the relationship between the number of bedroom screen devices and children's EBDs, as well as between the age of first screen use and EBDs, but not between hours of screen use and EBDs. Controlling for age, developmental level, and family income, children who started using screens earlier than 18 months and who had screen devices in their bedrooms had significantly more sleep problems and EBDs than those without., Conclusion: Children with neurodevelopmental conditions may have more difficulties disengaging from screen devices in their bedrooms, and an earlier age of screen exposure may contribute to more chronic disruption of sleep.

A strategy to construct multivariate biomarkers for exposure to algal neurotoxins via correlating changes to the profiles of a series of neurotransmitters and their metabolites in the central nervous system (CNS) of exposed test organism is reported. 3-Month-old marine medaka (Oryzais melastigma) were exposed to waterborne brevetoxin PbTx-1 at two sub-lethal dose levels (0.5 and 2.5 μg-PbTx-1 L -1 ) for a duration of 12 h before quantification of 43 selected neurotransmitters and metabolites in their CNS were measured via dansyl chloride derivatization and LC-MS/MS determination. The profiling data were analyzed by multivariate statistical analyses, including principle component analysis (PCA), projection on latent structure-discriminate analysis (PLS-DA) and orthogonal projection on latent structure-discriminate analysis (OPLS-DA). Neurotransmitters and metabolites related to activation of voltage-gated sodium channels (VGSCs), N-methyl-D-aspartic acid receptors (NMDARs) and cholinergic neurotransmission were found to contribute significantly to class separation in the corresponding OPLS-DA models. Those models obtained from different exposure dosages were correlated by the Shared and Unique Structures Plot (SUS-plot) to identify appropriate variables for the construction of exposure biomarkers in the form of multivariate predictive scores. The established biomarkers for male and female medaka fish were able to predict acute sub-lethal exposure to PbTx-1 with good sensitivity and specificity (male fish: sensitivity 94.7%, specificity 80.0%; female fish: sensitivity 91.4%, specificity 83.3%). Neurotransmitter profiles in the CNS of medaka fish that should have recovered from exposure to PbTx-1 have also been determined to reveal long-term impacts to the CNS of the affected organism even after the exposure has been interrupted., (Copyright © 2019. Published by Elsevier B.V.)

Background: Individuals with bipolar disorder respond to affective symptoms with a range of coping behaviours, which may further maintain the symptoms., Aims: To examine moment-to-moment dynamics between affective states and coping behaviours, and to evaluate the role of cognitive appraisals of internal states as moderators., Method: Forty-six individuals with bipolar disorder completed a clinical interview and an experience sampling assessment over 6 days. Time-lagged analyses were conducted by multilevel regression modelling., Results: A total of 1807 momentary entries were analysed. Negative affect predicted an increase in rumination at the subsequent time point (β = 0.21, s.e. = 0.08, P = 0.009, 95% CI 0.05-0.36), and vice versa (β = 0.03, s.e. = 0.01, P = 0.009, 95% CI 0.01-0.05). Positive affect predicted an increase in adaptive coping (β = 0.26, s.e. = 0.11, P = 0.018, 95% CI 0.04-0.47), and vice versa (β = 0.02, s.e. = 0.01, P = 0.019, 95% CI 0.00-0.03). Positive affect also predicted a decrease in rumination (β = -0.15, s.e. = 0.06, P = 0.014, 95% CI -0.26 to -0.03), and vice versa (β = -0.03, s.e. = 0.01, P = 0.016, 95% CI -0.06 to -0.01). Extreme cognitive appraisals predicted stronger associations between affective states and coping behaviours., Conclusions: Feedback loops between affective states and coping behaviours were revealed in the daily life of individuals with bipolar disorder, which were moderated by extreme cognitive appraisals., Declaration of Interest: None.

A series of substituted 9-methylenylanthracene photocages for diphenylphosphinothioesters have been synthesized to explore their photo-uncaging properties by visible light. Substituents such as phenyl, p-trifluoromethylphenyl, p-methoxyphenyl, ethyn-1-ylbenzene, and 3,3-dimethylbut-1-yn-1-yl have been introduced in order to extend the π-conjugation of the photocage and to shift the wavelength response of the uncaging process to the visible spectral range. Among these new photocages, the (10-(3,3-dimethylbut-1-yn-1-yl)anthracen-9-yl)methyl has been shown to have the best performance in terms of fast photo-uncaging and minimal byproduct formation. It is responsive to both UV and visible photoexcitation. Quantum yields of the photoinduced heterolytic anthracenylmethyl-phosphorus bond cleavage at 366 and 416 nm were found to be 0.08 and 0.025, respectively. This photocage enables traceless Staudinger ligation to be triggered by photoirradiation in the visible spectral range for bioconjugation applications. We demonstrate this with a series of visible-light-induced oligopeptide syntheses via the conjugation of amino acid/oligopeptide building blocks by the characteristic peptide linkage attained by traceless Staudinger ligation. Yields of the resultant conjugated oligopeptides ranged from 31 to 43%. This new photocage opens up the possibility of in situ synthesis of functional proteins/peptides mediated by visible-light-induced photoclick processes for the regulation of cellular/metabolic functions of life systems.

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization., Competing Interests: The authors have declared that no competing interests exist.

Purpose: The purpose of this study was to describe an attempt to apply item-response theory (IRT) and the Rasch model to construction of speech-recognition tests. A set of word-recognition test items applicable to children as young as 3 years old-with any level of hearing sensitivity, with or without using hearing devices-was developed., Method: Test items were constructed through expert consultation and by reference to some established language corpora, validated with 121 participants with various degrees of hearing loss and 255 with typical hearing. IRT and the Rasch model were applied to evaluate item quality., Results: Eighty disyllabic word items were selected in accordance with IRT. The speech-recognition abilities of the 376 young participants are reported. The IRT analyses on this set of data are also discussed., Conclusions: A new set of speech-recognition test materials in Cantonese Chinese has been developed. Construction of short equivalent lists may be performed in accordance with IRT item qualities. Clinical applications of this test tool in the particular language population are discussed.

Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. pCOMB amplifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross-pair correlation analysis (cpCOMB) reveals that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics.

Background: Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear., Objective: To define the effect of inhibition of Src activity and expression on neuronal survival., Results: In agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt., Conclusions: Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment., Methods: We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo., Results: Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs., Conclusions: The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus., (© 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.)

The STAT3 signal transducer and activator of transcription is a key mediator of gene transcription in response to cytokines such as oncostatin M (OSM). We performed direct live cell imaging of GFP-tagged STAT3 proteins for the first time, showing transient relocalization of STAT3α to the nucleus following OSM exposure, in contrast to sustained nuclear relocalization of the shorter STAT3β spliceform. To explore this further, we applied fluorescence recovery after photobleaching (FRAP) to determine the nuclear import kinetics of STAT3α and β, as well as of a C-terminal truncation derivative STAT3ΔC comprising only the sequence shared by the spliceforms, in the absence or presence of OSM. The rates of basal nuclear import for STAT3β and STAT3ΔC were significantly faster than those for STAT3α. Strikingly, OSM slowed the import rates of all the three STAT3 proteins, whereas the import rates of GFP alone or a classical importin-mediated cargo were unaffected, with analysis of Y705F mutant derivatives for all the three STAT3 constructs, or of a S727A mutant within the unique C-terminus of STAT3α, reinforcing the contribution of specific phosphorylation to the cytokine-stimulated changes. The results introduce a new paradigm where cytokine treatment prolongs nuclear retention simultaneous with decreasing rather than increasing the rate of nuclear import., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Persistent STAT3 phosphorylation and nuclear retention are hallmarks of a range of pathologies suggesting the importance of STAT3 transcriptional responses in disease progression. Since hyperosmotic stress (HOS) is a hallmark of diseases such as diabetes and asthma, we analysed the impact of HOS on cytokine-stimulated STAT3 signalling. In contrast to transient STAT3 Y705 and S727 phosphorylation in murine embryonic fibroblasts (MEFs) stimulated by the interleukin-6 family cytokine, leukemia inhibitory factor (LIF), under non-stress conditions, HOS induced by sorbitol treatment increased STAT3 S727 but not Y705 phosphorylation. Strikingly, combined LIF+HOS treatment stimulated persistent STAT3 Y705 and S727 phosphorylation and nuclear localisation, but STAT3 nuclear accumulation was slowed during HOS, likely reflecting the mislocalisation of Ran and importin-α3 during HOS that also reduced the nuclear localisation of classical importin-α/β-recognised nuclear import cargoes. Strikingly, combined LIF+HOS exposure, even though stimulating STAT3 phosphorylation and nuclear accumulation did not elicit a transcriptional output, as demonstrated by qPCR analyses of its target genes SOCS3 and c-Fos. Our analysis thus shows for the first time that HOS can disconnect nuclear, phosphorylated STAT3 from transcriptional outcomes, and emphasizes the importance of assessing STAT3 target gene changes in addition to STAT3 phosphorylation status and localisation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although cytokine-driven STAT3 phosphorylation and activation are often transient, persistent activation of STAT3 is a hallmark of a range of pathologies and underpins altered transcriptional responses. As triggers in disease frequently include combined increases in inflammatory cytokine and reactive oxygen species levels, we report here how oxidative stress impacts on cytokine-driven STAT3 signal transduction events. In the model system of murine embryonic fibroblasts (MEFs), combined treatment with the interleukin-6 family cytokine Leukemia Inhibitory Factor (LIF) and hydrogen peroxide (H2O2) drove persistent STAT3 phosphorylation whereas STAT3 phosphorylation increased only transiently in response to LIF alone and was not increased by H2O2 alone. Surprisingly, increases in transcript levels of the direct STAT3 gene target SOCS3 were delayed during the combined LIF + H2O2 treatment, leading us to probe the impact of oxidative stress on STAT3 regulatory events. Indeed, LIF + H2O2 prolonged JAK activation, delayed STAT3 nuclear localisation, and caused relocalisation of nuclear STAT3 phosphatase TC-PTP (TC45) to the cytoplasm. In exploring the nuclear import/ export pathways, we observed disruption of nuclear/cytoplasmic distributions of Ran and importin-alpha3 in cells exposed to H2O2 and the resultant reduced nuclear trafficking of Classical importin-alpha/3-dependent protein cargoes. CRM1-mediated nuclear export persisted despite the oxidative stress insult, with sustained STAT3 Y705 phosphorylation enhancing STAT3 nuclear residency. Our studies thus reveal for the first time the striking impact of oxidative stress to sustain STAT3 phosphorylation and nuclear retention following disruption of multiple regulatory events, with significant implications for STAT3 function.

The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress., (© 2013.)

Aims: We explore the role of an elevated O2(-):H2O2 ratio as a prosurvival signal in glioma-propagating cells (GPCs). We hypothesize that depleting this ratio sensitizes GPCs to apoptotic triggers., Results: We observed that an elevated O2(-):H2O2 ratio conferred enhanced resistance in GPCs, and depletion of this ratio by pharmacological and genetic methods sensitized cells to apoptotic triggers. We established the reactive oxygen species (ROS) Index as a quantitative measure of a normalized O2(-):H2O2 ratio and determined its utility in predicting chemosensitivity. Importantly, mice implanted with GPCs of a reduced ROS Index demonstrated extended survival. Analysis of tumor sections revealed effective targeting of complementarity determinant 133 (CD133)- and nestin-expressing neural precursors. Further, we established the Connectivity Map to interrogate a gene signature derived from a varied ROS Index for the patterns of association with individual patient gene expression in four clinical databases. We showed that patients with a reduced ROS Index demonstrate better survival. These data provide clinical evidence for the viability of our O2(-):H2O2-mediated chemosensitivity profiles., Innovation and Conclusion: Gliomas are notoriously recurrent and highly infiltrative, and have been shown to arise from stem-like cells. We implicate an elevated O2(-):H2O2 ratio as a prosurvival signal in GPC self-renewal and proliferation. The ROS Index provides quantification of O2(-):H2O2-mediated chemosensitivity, an advancement in a previously qualitative field. Intriguingly, glioma patients with a reduced ROS Index correlate with longer survival and the Proneural molecular classification, a feature frequently associated with tumors of better prognosis. These data emphasize the feasibility of manipulating the O2(-):H2O2 ratio as a therapeutic strategy.

p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.

Unlabelled: We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay., Importance: The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.

The 38-residue SBP-Tag binds to streptavidin more tightly (K(d) -/= 2.5-4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12-14) and a C-terminal HPQ sequence (residues 31-33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17-28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1-10 and 35-38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11-34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag-streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

Background: Sequence-independent amplification of clinical specimens can lead to the identification of novel pathogens., Objectives: To identify novel viruses in human stool specimens from patients with diarrhea and to investigate the ecology and clinical significance of such viruses., Study Design: Nucleic acid extracted from stool specimens from patients with diarrhea with no known etiology were subjected to random PCR amplification and Roche/454 pyrosequencing. Novel viruses identified were genetically and epidemiologically characterized., Results: Four gyroviruses, chicken anemia virus (CAV), human gyrovirus (HGV)/avian gyrovirus 2 (AGV2), gyrovirus 3 (GyV3) and a novel gyrovirus (tentatively designated as gyrovirus 4 (GyV4)) were identified in human stool specimens. GyV4, as well as CAV and AGV2/HGV were also detected in chicken skin and meat used for human consumption., Conclusions: A novel gyrovirus (GyV4) was identified in human stool and in chicken meat sold for human consumption. This virus was phylogenetically distinct from previously reported gyroviruses in chicken and humans (chicken anemia virus, human gyrovirus, avian gyrovirus 2 and recently reported gyrovirus 3). The epidemiology and pathogenesis of this virus in humans and in chicken needs to be further investigated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3β spliceform was initially described as a negative regulator of STAT3α, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3α and STAT3β at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3β following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3α. Importantly, co-expression of the spliceforms revealed that STAT3β enhanced and prolonged the phosphorylation and nuclear retention of STAT3α, but a STAT3β R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3α. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3α or STAT3β, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3β-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3β as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3α phosphorylation and nuclear retention after cytokine stimulation.

Objectives: This study aimed to evaluate the benefits of ClearVoice strategy on speech perception in noise and in everyday listening situations in Cantonese-speaking cochlear implant users., Methods: Twelve experienced adult users of the Harmony implant and HiRes 120 sound processing participated in the study. The study employed a prospective within-subjects design wherein speech recognition in adults using HiRes 120 without ClearVoice turned on (control option) was compared to their performance with HiRes 120 with ClearVoice turned on. Each subject was evaluated with two different ClearVoice gain settings: -12 dB (ClearVoice medium) and -18 dB (ClearVoice high) after one-week of use. The Cantonese hearing in noise test and a questionnaire were used as the outcome measures., Results: Subjects performed significantly better with ClearVoice medium than with control option in noise. No significant difference in performance was noted among the 3 settings in quiet. Most subjects reported high level of satisfaction with ClearVoice in daily listening situations and preferred to keep ClearVoice on., Conclusion: ClearVoice can help cochlear implant recipients to hear better in noise.

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

Background: Bolus nasogastric tube feeding is common. Unsafe practices such as failure to confirm tube placement can result in death. It is vital to ensure that nurses are adopting safe practices., Objective: To evaluate nurses' practices on bolus nasogastric feeding relating to verification of tube placement, management of gastric residual volume, and response to complications during feeding., Design: Cross-sectional, self-administered survey using clinical scenarios., Setting and Participants: All nurses who worked in the general wards in a tertiary hospital in August 2008., Methods: We developed six clinical scenarios to describe common clinical situations in nurses' daily practices. Participants were instructed to choose the responses that best reflected their practices, and to return the completed questionnaires to the study member present., Results: The survey participation rate was 99.5% (1203 nurses). Seventy-six percent would choose two or more methods to verify placement when they were in doubt. Percentage of hydrogen (pH) testing was the most common first method of checking tube placement. The second and third self-reported methods were auscultation and the bubble test. Few chose radiography to confirm tube placement. When the aspirate was pH 7, and in the presence of positive auscultation, most participants would take further steps to confirm placement. There were variations in the nurses' responses on managing the gastric residual volume, with 78.1% indicating that they would return the aspirate. Most nurses lacked the knowledge to effectively manage patients' distress during tube feeding., Conclusions: The findings showed that the majority of participants reported that they would exercise due caution by taking additional measures to check tube placement when in doubt. The practice gaps identified in the study highlighted a need to realign our care to best practices. Following the study, we revised the institution's guideline, reinforced specific safety precautions on nasogastric feeding, and incorporated clinical scenarios in our training., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus-infected cells and inhibit viral replication in vitro. In Rag2(-/-)γc(-/-) immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus-infected Rag2(-/-)γc(-/-) mice reconstituted with Vγ9Vδ2 T cell-depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget's disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza.

In introductory biochemistry courses, it is often a struggle to teach the basic concepts of acid-base chemistry in a manner that is relevant to biological systems. To help students gain a more intuitive and visual understanding of abstract acid-base concepts, a simple graphical construct called the pH ruler Java applet was developed. The applet allows students to visualize the abundance of different protonation states of diprotic and triprotic amino acids at different pH values. Using the applet, the student can drag a widget on a slider bar to change the pH and observe in real time changes in the abundance of different ionization states of this amino acid. This tool provides a means for developing more complex inquiry-based, active-learning exercises to teach more advanced topics of biochemistry, such as protein purification, protein structure and enzyme mechanism.

Background: Oral hygiene is necessary to maintain the health and well-being of hospitalised patients. However, studies indicated that nurses lacked the evidence-based knowledge to deliver proper care. A prior survey conducted on nurses working in the intensive care and high dependency units in our institution revealed that oral care practices varied and highlighted the need for an oral care protocol. These triggered us to embark on an evidence-based project on oral care to improve patients' oral health during their hospitalisation., Aim: We aimed to translate the best available evidence to improve our oral care practices., Methods: We followed the Iowa Model of Evidence-based Practice. Having identified a problem, we formed a team. We searched for best available evidence on oral care management and appraised them using the Scottish Intercollegiate Guidelines Network checklists. We developed an evidence-based oral care protocol and outlined key changes necessary to improve patients' oral care during hospitalisation. We chose the medical intensive care unit and a neurology ward to pilot the changes. The changes included introducing an oral assessment guide to assess patients' oral condition and intervening appropriately according to their risk levels, recommending 0.2% chlorhexidine solution as the main oral cleaning solution, and standardising oral care documentation. We compared the pre- and post-audits to assess improvement in the nurses' knowledge and compliance to the protocol. Auditors also physically inspected the oral health of functionally dependent patients under the charge of the audited nurses., Results: The team involved 25 patients and 25 nurses in each pre- and post-audits. The median pre- and post-scores were 60% and 100%, respectively. The results revealed that the project led to improvements in nurses' oral care knowledge and practices, especially in nurses taking appropriate interventions as recommended, and in correctly using oral care products to clean patients' mouths. Auditors reported improvement in patients' oral care during their physical assessments of patients' oral cavities. This finding was supported by feedback from other healthcare providers., Conclusions: This evidence-based project heightened nurses' awareness and knowledge on oral care, and led to nurses providing practices based on best available evidence. These have translated to improvements in patients' oral health., (© 2011 The Authors. International Journal of Evidence-Based Healthcare © 2011 The Joanna Briggs Institute.)

Background: Several different procedures have been advocated for thoracic spine dumbbell tumor resection, combining thoracic and neurosurgical approaches, in single and multiple stages, using various incisions and positions. These have led to controversies in the ideal management. The authors report their analysis of a series of 11 patients successfully treated through a one-step midline approach for complete resection and instrumentation when indicated under intraoperative CT (ICT) guidance., Methods: The patients' clinical presentations, imaging results, operative findings and follow-up were reviewed in 11 patients (age ranged from 11 to 62 years), over the period from August 2007 to May 2010. A single-stage, posterior midline incision approach with laminectomy, facetectomy, costotransversectomy, for complete resection of intraspinal and paraspinal components of tumor was used. Spinal instrumentation under ICT guidance was also carried out in relevant (six) cases with tumors involving junctional spinal regions such the cervico-thoracic or thoraco-lumbar region., Results: The initial clinical presenting symptom was pain in eight patients and paresthesia in one, while two patients were detected incidentally on routine chest X-rays. Total excision was achieved in 10 patients (9 schwanommas, 1 neurofibroma) with the exception of one patient who had a recurrent malignant peripheral nerve sheath tumor adherent to the vertebral artery. No significant postoperative complications occurred and an early mobilization/discharge was achieved in all patients with an average hospital stay of 5 days., Conclusions: A one-step approach through a posterior midline incision is feasible, safe and efficient for complete excision of thoracic dumbbell tumors. This approach facilitates laminectomy, facetectomy, costotransversectomy and instrumentation under ICT guidance, while limiting muscle damage, blood loss, operative time, postoperative pain, thus enabling early mobilization with a reduced hospital stay.

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.

The JNKs (c-Jun N-terminal kinases) are stress-activated serine/threonine kinases that can regulate both cell death and cell proliferation. We have developed a cell system to control JNK re-expression at physiological levels in JNK1/2-null MEFs (murine embryonic fibroblasts). JNK re-expression restored basal and stress-activated phosphorylation of the c-Jun transcription factor and attenuated cellular proliferation with increased cells in G1/S-phase of the cell cycle. To explore JNK actions to regulate cell proliferation, we evaluated a role for the cytosolic protein, STMN (stathmin)/Op18 (oncoprotein 18). STMN, up-regulated in a range of cancer types, plays a crucial role in the control of cell division through its regulation of microtubule dynamics of the mitotic spindle. In JNK1/2-null or c-Jun-null MEFs or cells treated with c-Jun siRNA (small interfering RNA), STMN levels were significantly increased. Furthermore, a requirement for JNK/cJun signalling was demonstrated by expression of wild-type c-Jun, but not a phosphorylation-defective c-Jun mutant, being sufficient to down-regulate STMN. Critically, shRNA (small hairpin RNA)-directed STMN down-regulation in JNK1/2-null MEFs attenuated proliferation. Thus JNK/c-Jun regulation of STMN levels provides a novel pathway in regulation of cell proliferation with important implications for understanding the actions of JNK as a physiological regulator of the cell cycle and tumour suppressor protein.

Spinal perimedullary arteriovenous fistula (AVF) or dural arteriovenous fistula (DAVF) presenting as intracranial subarachnoid haemorrhage (SAH) is uncommon. A total of 16 cases have been reported to date. A majority of the reports described cervical spinal DAVF, while two other case reports described intracranial SAH secondary to lumbar and thoracic DAVF, respectively. We report a 61-year-old Chinese man with intracranial SAH secondary to thoracic DAVF aneurysm, who presented with sudden, severe chest pain, initially suggestive of aortic dissection/acute myocardial infarction. However, a careful examination of the history and physical signs, followed by appropriate and timely investigations enabled effective treatment to be administered promptly with a good outcome. This serves to illustrate the importance of investigating the entire cerebrospinal system when neurological symptoms and clinical signs suggest extracranial primary pathology.

Background: Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants., Results: The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus., Conclusions: The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.

Introduction: Functional magnetic resonance imaging (fMRI) is a neuroradiological technique for the localisation of cortical function. FMRI made its debut in cognitive neuroscience and then eventually to other clinical applications. We report our experience with pre-surgical fMRI on a high field scanner, based purely on a clinical platform., Materials and Methods: The protocols included motor, auditory, visual and language fMRI. The choice of protocols was dependant on clinical request and lesion locale., Results: Retrospective analysis and audit of the fi rst 30 consecutive patients over a 12-month period revealed that about 85% of patients had a successful examination. In a pictorial essay, we demonstrate that patients with weakness in performing a motor task showed abnormal activations of the pre-motor and supplementary motor areas., Conclusion: FMRI data greatly enhances the pre-surgical planning process and the conduct of surgery when it is incorporated into the surgical navigation system in the operating theatre.

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-alpha, IFN-beta, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease. Here we show that TNF-alpha, IFN-beta, and IFN-lambda1 are the key mediators directly induced by the H5N1 virus in primary human macrophages. In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3). IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-beta, IFN-lambda1, and MCP-1 but only to a partial reduction of TNF-alpha. IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus. We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-beta, IFN-lambda1, and MCP-1 but only partly control TNF-alpha induction. A more precise identification of the differences in the regulation of TNF-alpha and IFN-beta could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.

Objective: This study aimed at developing a theoretically driven open-set speech recognition test for pediatric clinical population of cochlear implant and/or hearing aid users, with Cantonese Chinese as their first language, to track progress in speech recognition performance as an outcome measurement of their rehabilitation., Methods: Six monosyllabic and six disyllabic word lists were generated from the Cantonese CHILDES language database, constructed according to the Neighborhood Activation Model. There were three lexically "easy" and three lexically "hard" word lists in each sub-test, with 25 items in each list. Four pediatric cochlear implant users and 10 hearing aid users, with bilateral congenital severe to profound sensorineural hearing impairment and below the age of 10, participated in the study. Their performances on word recognition and phoneme recognition with the new test lists, as well as the inter-list equivalency, inter-rater reliability, and face validity of the new materials, were investigated., Results: Word recognition was higher among disyllables than monosyllables. Lexically "easy" disyllabic words were better recognized than their "hard" counterparts and the monosyllables. No significant difference was noted among the three lists in each combination of syllable structure and lexical property. High inter-rater reliability, as well as high correlation between Cantonese LNT score and a receptive vocabulary test score, were revealed., Conclusions: These newly developed test lists provided reliable information on spoken word recognition of pediatric hearing prosthesis users with severe to profound hearing impairment. Inter-list equivalency and inter-rater reliability allowed monitoring of rehabilitation progress on such specific pediatric clinical population with this new test. (255).

Objectives: Universal infant hearing screening using otoacoustic emission and auditory brain-stem response audiometry is widely administered to attain the goals of early identification of, and intervention for hearing impairment. Concerns regarding screening specificity have, however, been raised. False positives may result from vernix occlusion in the ear canal or transient middle ear effusion, and can result in substantial costs to health care systems. The current study investigates the effects of age and time interval between tests on hearing assessment results., Setting & Participants: Three hundred and seventeen positive screens from a 2-stage distortion product otoacoustic emission (DPOAE) screening programme in Hong Kong, who subsequently received diagnostic auditory brainstem response (ABR) assessment and monitoring, were investigated., Main Outcome Measures: Differences in diagnostic ABR results were compared among infants of different ages at tests, and with different time lapses after DPOAE screening. The proportion of those having persistent hearing impairment, conductive loss and impairment of moderate degree or above, were also compared., Results: A significantly higher rate of normal ABR thresholds (60%versus 24%) was noted in infants assessed after age 50 days, and in infants diagnostically assessed with a time lapse of over 20 days post-DPOAE screening (65%versus 42%)., Conclusions: Delaying diagnostic ABR assessment may reveal a higher percentage of normal thresholds, and hence probably higher specificity. Time delay may allow for spontaneous resolution of transient outer and middle ear conditions. However, the goals of early identification and intervention, as well as possible parental anxiety with delayed assessment, should also be considered when reviewing infant hearing screening schedules.

This was a pilot study to compare the cerebral haemodynamics and neurochemical changes in patients with primary basal ganglia haemorrhage (PBGH), who underwent conventional blood glucose level (BGL) control and intensive BGL control with continuous titrated insulin therapy. Patients admitted over an 18-month period with PBGH after evacuation of haematoma were retrospectively divided into two groups according to the method used for BGL control: the 'intensive' group consisted of patients who underwent continuous titrated insulin infusion to maintain a lower normoglycemic level of 4-8 mmol/L, and the 'conventional' group consisted of patients whose BGL was maintained at between 8.1 and 10.0 mmol/L using conventional 'sliding scale' bolus subcutaneous insulin administration. Data on cerebral haemodynamics, interstitial brain oxygenation (PtiO(2)) and neurochemical monitoring were collected via microcatheters inserted in the perihaemorrhagic penumbral region. A homogenous group of 12 patients with haemorrhage originating in the deep basal nuclei was identified. Five patients (42%) were included in the intensive group, and seven patients (58%) were included in the conventional group. The mean intracranial pressure, mean arterial pressure, BGL, extracellular (EC) lactate, EC glutamate, EC pyruvate and EC glycerol levels and the lactate/pyruvate ratio were found to be significantly lower (p<0.001) in the intensive group compared with the conventional group, but the mean PtiO(2) and amount of insulin administered were higher (p<0.001) in the intensive group. The mean cerebral perfusion pressure and EC glucose did not differ significantly between the two groups of patients. Maintenance of lower normoglycaemia (4-8 mmol/L) with continuous titrated insulin therapy is associated with improved cerebral haemodynamics, oxygenation and neurochemistry in the perihaemorrhagic penumbral region.

Objective: There is well-documented evidence in the literature concerning a high prevalence of deafness in children with Down syndrome (DS). The aim of this study was to examine the extent of hearing impairment and address the rehabilitation needs of a Chinese population with DS who were either in special schools or integrated into mainstream schools., Methods: This study screened 92 children with DS at their own schools and 11 were reassessed in the University of Hong Kong Hearing Centre. Hearing status of the children with DS was analyzed on the basis of their screening and reassessment results for tympanometric, transient evoked otoacoustic emission (TEOAE) and pure tone audiometric examinations., Results: A high point prevalence of hearing impairment (78% by ears or 90% by participants) in a Chinese school-aged sample of children with DS was noted. The most common degree of loss was mild to moderate. No significant gender difference, age effect or ear asymmetry was found for tympanometric failure or absence of TEOAE. An unfavorable mean speech intelligibility index score (0.2) was found for this group of children., Conclusions: Sound field amplification and suitable acoustic modifications to classrooms were recommended for Chinese children with DS in Hong Kong to improve their listening and learning environment. The point prevalence of hearing impairment in older children with DS in this study was in contrast to a previous local study on a younger age group. Further effort is needed to determine the role of possible aging effects on the type and prevalence of hearing impairment in populations with DS.

In 1997, avian influenza virus H5N1 was transmitted directly from chicken to human and resulted in a severe disease that had a higher mortality rate in adults than in children. The characteristic mononuclear leukocyte infiltration in the lung and the high inflammatory response in H5N1 infection prompted us to compare the chemokine responses between influenza virus-infected adult and neonatal monocyte-derived macrophages (MDMs). The effects of avian influenza virus A/Hong Kong/483/97 (H5N1) (H5N1/97), its precursor A/Quail/Hong Kong/G1/97 (H9N2) (H9N2/G1), and human influenza virus A/Hong Kong/54/98 (H1N1) (H1N1/98) were compared. Significantly higher expression of CCL2, CCL3, CCL5, and CXCL10 was induced by avian influenza viruses than by human influenza virus. Moreover, the increase in CCL3 expression in H5N1/97-infected adult MDMs was significantly higher than that in neonatal MDMs. Enhanced expression of CCR1 and CCR5 was found in avian virus-infected adult MDMs. The strong induction of chemokines and their receptors by avian influenza viruses, particularly in adult MDMs, may account for the severity of H5N1 disease.