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SummaryDiagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to prevent potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both non-infectious virus-like particles (VLPs) and soluble non-structural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both VLP- and NS1-MAC-ELISAs were found to have similar sensitivity for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera. Group cross reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher P/N values than homologous wild-type VLPs. Therefore, FP-VLPs were used to develop the algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80% with no statistical significance. A novel approach to differentiate ZIKV from DENV infection serologically has been developed. The accuracy can reach up to 95% when combining both VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.

Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to mitigate potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both noninfectious wild-type (wt) virus-like particles (VLPs) and soluble nonstructural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both ZIKV-derived wt-VLP- and NS1-MAC-ELISAs were found to have similar sensitivities for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera, although lower cross-reactivity to DENV2/3-NS1 was observed. Furthermore, group cross-reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher positive-to-negative values than homologous wt-VLPs. Therefore, we used DENV-2/3 and ZIKV FP-VLPs to develop a novel, serological algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80%, with no statistical significance. The accuracy can reach up to 95% with the combination of FP-VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.

© 2018 Copyright held by the owner/author(s). Machine learning approaches have been effective in predicting adverse outcomes in different clinical settings. These models are often developed and evaluated on datasets with heterogeneous patient populations. However, good predictive performance on the aggregate population does not imply good performance for specific groups. In this work, we present a two-step framework to 1) learn relevant patient subgroups, and 2) predict an outcome for separate patient populations in a multi-task framework, where each population is a separate task. We demonstrate how to discover relevant groups in an unsupervised way with a sequence-to-sequence autoencoder. We show that using these groups in a multi-task framework leads to better predictive performance of in-hospital mortality both across groups and overall. We also highlight the need for more granular evaluation of performance when dealing with heterogeneous populations.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein we show for the first time that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our results revealed that a “epitope-resurfaced” mature-form dengue VLP has the potential to induce quaternary structure-recognizing broad CR NtAbs.

Swine are a critical amplifying host involved in human Japanese encephalitis (JE) outbreaks. Cross-genotypic immunogenicity and sterile protection are important for the current genotype III (GIII) virus-derived vaccines in swine, especially now that emerging genotype I (GI) JE virus (JEV) has replaced GIII virus as the dominant strain. Herein, we aimed to develop a system to generate GI JEV virus-like particles (VLPs) and evaluate the immunogenicity and protection of the GI vaccine candidate in mice and specific pathogen-free swine. A CHO-heparan sulfate-deficient (CHO-HS(-)) cell clone, named 51-10 clone, stably expressing GI-JEV VLP was selected and continually secreted GI VLPs without signs of cell fusion. 51-10 VLPs formed a homogeneously empty-particle morphology and exhibited similar antigenic activity as GI virus. GI VLP-immunized mice showed balanced cross-neutralizing antibody titers against GI to GIV viruses (50% focus-reduction micro-neutralization assay titers 71 to 240) as well as potent protection against GI or GIII virus infection. GI VLP-immunized swine challenged with GI or GIII viruses showed no fever, viremia, or viral RNA in tonsils, lymph nodes, and brains as compared with phosphate buffered saline-immunized swine. We thus conclude GI VLPs can provide sterile protection against GI and GIII viruses in swine.

The maintenance of upright posture involves constant adjustment to external and internal perturbations. This balancing act is often assumed to be an automatic process, but studies suggest that cognitive processes, particularly attention, are necessary for the control of posture. The current study examines the role of attention in balance using a dual-task paradigm. Twenty-four healthy young adults performed a sit-to-stand (STS) task on either a stable or unstable platform while performing a secondary cognitive task of counting backwards aloud. Movement of the upper and lower body was analyzed using the largest Lyapunov exponent (λ1) and standard deviation (SD). Results replicated earlier findings (Gibbons, Amazeen, & Likens, 2018) that the transition from sit to stand was marked by increased variability and a temporary destabilization of postural control. Participants exhibited greater movement variability overall on the unstable platform (large SD), but small λ1 indicated that movement was controlled. During second task performance, SD increased for the upper body only. Further research is necessary to understand the interaction between attention and balance in young adults.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. The vaccine candidates under development require a tetravalent immunogen to induce a balanced immunity against all four serotypes of dengue virus. Herein we show that mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher and broader neutralization antibodies (NtAbs) against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. The cryo-electron microscopy reconstruction showed that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Most importantly, maternally transferred antibodies derived from mD2VLP-vaccinated female mice protected suckling mice from lethal challenge by all four serotypes of DENV. Our results support the fact that a universal dengue vaccine that protects against all four serotypes of dengue viruses can be achieved by using an immunogen such as mD2VLP.

Background: Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to prevent potential severe health outcomes. Methods: A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both non-infectious virus-like particles (VLPs) and soluble non-structural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Findings: Both VLP- and NS1-MAC-ELISAs were found to have similar sensitivity for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera. Group cross reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher P/N values than homologous wild-type VLPs. Therefore, FP-VLPs were used to develop the algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80% with no statistical significance. Interpretation: A novel approach to differentiate ZIKV from DENV infection serologically has been developed. The accuracy can reach up to 95% when combining both VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist. Funding Statement: Portions of this publication was made possible through support provided by the CDC intramural research fund and the Office of Infectious Disease, Bureau for Global Health, U.S. Agency for International Development, under the terms of an Interagency Agreement with CDC. The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of the CDC and U.S. Agency for International Development. Declaration of Interests: We declare no competing interests. Ethics Approval Statement: This study was conducted and reported in accordance with the Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. Informed consent documents for all eligible participants were waived based on the protocol number 6874. An institutional review board (IRB) waiver to use this serum panel for research purposes was approved by the CDC-human studies review board.

Machine learning approaches have been effective in predicting adverse outcomes in different clinical settings. These models are often developed and evaluated on datasets with heterogeneous patient populations. However, good predictive performance on the aggregate population does not imply good performance for specific groups. In this work, we present a two-step framework to 1) learn relevant patient subgroups, and 2) predict an outcome for separate patient populations in a multi-task framework, where each population is a separate task. We demonstrate how to discover relevant groups in an unsupervised way with a sequence-to-sequence autoencoder. We show that using these groups in a multi-task framework leads to better predictive performance of in-hospital mortality both across groups and overall. We also highlight the need for more granular evaluation of performance when dealing with heterogeneous populations., Comment: KDD 2018

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.

Background Knowing the high and low peaks in comfort during a flight could be useful in prioritizing aircraft interior improvements. Objective The first objective of this study was to identify whether there are differences in comfort experiences during different phases of a flight. The second objective of this study was to identify similarities between recalled and real time reported comfort experiences. Methods 149 participants were asked to rate the comfort in the different phases of their last flight on a scale from 1-10. Additionally, a combination of a self-reporting design probe and generative interview was used to investigate the appraisal patterns of emotions in nine passengers. Results The 149 subjects reported the highest comfort after take-off and arriving at the destination, the lowest while stowing the luggage and during the cruise flight. The qualitative long haul inflight study showed after take-off and while arriving at the destination the most positive emotions and during the cruise flight there is a negative experience phase. Conclusions Suggestions are given to improve the cruise flight phase, by for example stimulation of movement or better service.

Background We have detected intramucosal bacteria within the sinus mucosa of patients with chronic rhinosinusitis (CRS), but our attempts at characterizing these did not yield any discernible genotypic or phenotypic differences from surface bacteria. We hypothesized that the presence of intramucosal microcolonies reflected host mucosal immune dysfunction. This study characterizes the activation status of T cells, B cells, and macrophages in the sinus mucosa of patients with CRS and controls and determines the impact of bacteria on mucosal immunology. Methods Swabs and mucosal biopsy specimens were taken from 27 patients with CRS undergoing sinus surgery and 9 patients with normal sinuses having transnasal pituitary surgery. Microcolonies were detected using Gram staining, and the immune cells were characterized by immunohistochemical techniques. Results Swab culture rates for Staphylococcus aureus were similar between CRS and controls. However, there were significantly more intramucosal microcolonies in CRS (59% versus 11%) than in controls (p = 0.02). There were significantly more immune cells in CRS. Percentage of activated T and B cells were similar between CRS and controls, but there were significantly more CD163+ M2 macrophages in patients with CRS (p = 0.0004). Furthermore, percentage of CD163+ macrophages showed a positive correlation with disease severity. The presence of bacteria had no impact on immunology or disease severity. Conclusion Tolerance of intramucosal microcolonies in CRS may reflect altered macrophage function in the host mucosa. The clinical severity of CRS is also dependent on the host mucosa immune dysfunction, rather than the presence of intramucosal microcolonies.

Designing a randomized controlled trial (RCT) to investigate a survival benefit in a rare disease such as pulmonary arterial hypertension (PAH) has considerable logistical and ethical constraints. In the SERAPHIN RCT, a 23% non-significant reduction in the risk of all-cause mortality was observed with macitentan 10mg vs placebo. As SERAPHIN enrolled patients in the same time frame as the US REVEAL registry, a prediction model based on REVEAL data was used to further explore the effect of macitentan on mortality. From REVEAL (N=3515), 1013 patients who would have met SERAPHIN eligibility criteria were selected (REVEAL analysis cohort [RAC]). Using the RAC, a prediction model for time to death up to 3 years was constructed based on 10 baseline prognostic variables. The model was used to predict survival of each of the 742 SERAPHIN patients had they received real-world treatment in the RAC. The average temporal profile of these patients was then compared with the observed survival of the macitentan 10mg group (n=242) using a log-rank test and Cox’s proportional hazard model. Over 3 years, the risk of mortality observed with macitentan 10mg was 31% lower than that predicted (p=0.033) (Fig). Real-world observational data can complement RCT data to provide a means of evaluating survival benefits in rare diseases. Merits and limitations of this approach will be discussed.

Existing machine learning methods typically assume consistency in how semantically equivalent information is encoded. However, the way information is recorded in databases differs across institutions and over time, often rendering potentially useful data obsolescent. To address this problem, we map database-specific representations of information to a shared set of semantic concepts, thus allowing models to be built from or transition across different databases. We demonstrate our method on machine learning models developed in a healthcare setting. In particular, we evaluate our method using two different intensive care unit (ICU) databases and on two clinically relevant tasks, in-hospital mortality and prolonged length of stay. For both outcomes, a feature representation mapping EHR-specific events to a shared set of clinical concepts yields better results than using EHR-specific events alone.

Fundamental understanding of structure-property relationships in hierarchically organized nanostructures is crucial for the development of new functionality, yet quantifying structure across multiple length scales is challenging. In this work, we used nondestructive X-ray scattering to quantitatively map the multiscale structure of hierarchically self-organized carbon nanotube (CNT) "forests" across 4 orders of magnitude in length scale, from 2.0 Å to 1.5 μm. Fully resolved structural features include the graphitic honeycomb lattice and interlayer walls (atomic), CNT diameter (nano), as well as the greater CNT ensemble (meso) and large corrugations (micro). Correlating orientational order across hierarchical levels revealed a cascading decrease as we probed finer structural feature sizes with enhanced sensitivity to small-scale disorder. Furthermore, we established qualitative relationships for single-, few-, and multiwall CNT forest characteristics, showing that multiscale orientational order is directly correlated with number density spanning 10

In mid-2015, Salvador, Brazil, reported an outbreak of Guillain-Barré syndrome (GBS), coinciding with the introduction and spread of Zika virus (ZIKV). We found that GBS incidence during April–July 2015 among those ≥12 years of age was 5.6 cases/100,000 population/year and increased markedly with increasing age to 14.7 among those ≥60 years of age. We conducted interviews with 41 case-patients and 85 neighborhood controls and found no differences in demographics or exposures prior to GBS-symptom onset. A higher proportion of case-patients (83%) compared to controls (21%) reported an antecedent illness (OR 18.1, CI 6.9–47.5), most commonly characterized by rash, headache, fever, and myalgias, within a median of 8 days prior to GBS onset. Our investigation confirmed an outbreak of GBS, particularly in older adults, that was strongly associated with Zika-like illness and geo-temporally associated with ZIKV transmission, suggesting that ZIKV may result in severe neurologic complications., Author summary Shortly following the introduction of Zika virus (ZIKV), a type of flavivirus transmitted by mosquitoes, into Brazil in early 2015, the Brazil Ministry of Health began receiving increased reports of a paralyzing condition known as Guillain-Barré syndrome (GBS). The areas with the greatest number of GBS cases appeared to correlate geographically and temporally with the areas reporting the highest rate of ZIKV infections. This association had been previously observed during a ZIKV outbreak in French Polynesia, however, this had not been systematically examined in a case-control investigation for the ZIKV outbreak in South America. In this investigation, the authors found that the occurrence of GBS in the affected population was nearly four times higher than would be expected, and the risk for GBS was particularly elevated among older adults. GBS was associated with ZIKV-like symptoms and with a combination of ZIKV-like symptoms plus laboratory evidence of a recent flavivirus infection. Taken together, these findings provide strong support for and greater understanding of the link between ZIKV and GBS.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141high podoplanin+, CD90+, ICAM1+, and VCAM1+ but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68+ APCs. CD68+ APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

The Kokobera virus group comprises mosquito-borne flaviviruses that cluster together phylogenetically. These viruses are unique to Australia and Papua New Guinea, and have been associated with a mild polyarticular disease in humans. Recent isolation of genetically diverse viruses within this group has prompted analysis of their genetic and phenotypic relationships. Phylogenetic analysis based on complete ORF, the envelope gene or the NS5/3' untranslated region supported the separation of the group into distinct species: Kokobera virus (KOKV), Stratford virus, New Mapoon virus, MK7979 and TS5273. Virulence studies in 3-week-old mice also provided the first evidence that a member of the KOKV group (MK7979) was neuroinvasive after intraperitoneal inoculation. In this context, our recent detection of KOKV group-specific antibodies in horses in the field suggests that these viruses should be considered in the epidemiology of flavivirus encephalitis in Australia. Refereed/Peer-reviewed

Vegetation that becomes overtopped usually experiences a decrease in abundance or species richness. When an overtopping plant alters the physiognomy of the existing vegetation (e.g., trees invading a shrubland), ecosystem processes can also be dramatically altered. Worldwide, Monterey pine (Pinus radiata) cultivars have been planted in Mediterranean-like climates and are known to invade surrounding natural communities. Ecological impacts resulting from these invasions have been widely investigated; however, the effects from solitary pine trees on the vegetation they overtop are lacking. Furthermore, studies on the impact of P. radiata cultivars from the California floristic province, where P. radiata is native, do not exist. In coastal California, north of the present-day range of native P. radiata stands, cultivars of this species have invaded northern coastal scrub vegetation. To determine the impact of pine invasion on species richness and structure in this habitat, floristic surveys were conducted in 20 blocks that consisted of invaded and uninvaded plots. An invaded plot contained two subplots located under the canopy of an isolated pine tree, whereas a paired, uninvaded plot contained two subplots located in coastal scrub adjacent to each pine. Pine trees selected ranged in size from 2.8 to 119 cm (1.1 to 46.9 in) basal diameter. Our results demonstrate that understory native cover and species richness are negatively correlated with tree size. Understory exotic plant cover and richness of species other than P. radiata did not show any correlation with tree size, mainly because exotic plants had a very low abundance overall.

Sampler filling time begins with the initial flash of blood in the needle hub until the preset sampler volume is obtained. Previous studies have shown statistically significant differences between arterial and venous sampler filling times, but included only a few subjects with abnormal blood pressures.To determine whether the time required to fill a vented arterial sampler is an accurate indication of a successful arterial blood sample in adults with abnormal blood pressure. We hypothesized that mean arterial pressure and arterial sampler filling time would have a negative correlation, and that venous sampler filling time would be significantly longer than arterial filling time.We studied 40 subjects: 25 arterial subjects, and 15 venous subjects. The arterial subjects included 3 groups: hypertensive, hypotensive, or normal. During the arterial and venipuncture procedures, we measured sampler filling time and recorded blood volume. The PO2 of the samples was measured. Additionally, blood pressure and SpO2 were measured in the arterial group.The mean sampler filling time was 220.4 s/mL for the venous group, and 18.1 s/mL for all 3 arterial groups combined. There were significant differences between each mean arterial sampler filling rate and mean venous filling rate (P.001). There were no significant differences in mean sampler filling rate between the arterial subgroups (P.99). The correlation between mean arterial pressure and filling rates was 0.06 (P = .38).There is a significant difference between arterial and venous filling rates. There was no relationship between filling time and abnormal mean arterial pressure. Regardless of the arterial pressure, the arterial sampler filling time can be used as an indicator of a successful arterial puncture at the bedside.

Developing quantitative descriptions of how stimulant and depressant drugs affect the respiratory system is an important focus in medical research. Respiratory variables—respiratory rate, tidal volume, and end tidal carbon dioxide—have prominent temporal dynamics that make it inappropriate to use standard hypothesis-testing methods that assume independent observations to assess the effects of these pharmacological agents. We present a polynomial signal plus autoregressive noise model for analysis of continuously recorded respiratory variables. We use a cyclic descent algorithm to maximize the conditional log likelihood of the parameters and the corrected Akaike’s information criterion to choose simultaneously the orders of the polynomial and the autoregressive models. In an analysis of respiratory rates recorded from anesthetized rats before and after administration of the respiratory stimulant methylphenidate, we use the model to construct within-animal z-tests of the drug effect that take account of the time-varying nature of the mean respiratory rate and the serial dependence in rate measurements. We correct for the effect of model lack-of-fit on our inferences by also computing bootstrap confidence intervals for the average difference in respiratory rate pre- and postmethylphenidate treatment. Our time-series modeling quantifies within each animal the substantial increase in mean respiratory rate and respiratory dynamics following methylphenidate administration. This paradigm can be readily adapted to analyze the dynamics of other respiratory variables before and after pharmacologic treatments.

Group and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs.

Background: The non-malignant cells of the tumour stroma have a critical role in tumour biology. Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention. For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts. Methods: Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts. Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively. Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression. Results: 5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts. Conclusion: 5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma. The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents.

Flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are among the most prevalent human disease-causing arboviruses world-wide. As they continue to expand their geographic range, multivalent flavivirus vaccines may become an important public health tool. Here we describe the immune kinetics of WNV DNA vaccination and the identification of a CD4 epitope that increases heterologous flavivirus vaccine immunogenicity. Lethal WNV challenge two days post-vaccination resulted in 90% protection with complete protection by four days, and was temporally associated with a rapid influx of activated CD4 T cells. CD4 T cells from WNV vaccinated mice could be stimulated from epitopic regions in the envelope protein transmembrane domain. Incorporation of this WNV epitope into DENV-2 DNA and virus-like particle vaccines significantly increased neutralizing antibody titers. Incorporating such potent epitopes into multivalent flavivirus vaccines could improve their immunogenicity and may help alleviate concerns of imbalanced immunity in multivalent vaccine approaches.