Motor vehicle crashes are a leading cause of injury death in the United States, and Memorial Day weekend is one of six holiday periods with an increased number of motor vehicle fatalities in the United States. However, few motor vehicle fatality comparisons were made between Memorial Day weekend and non-holiday periods. Our aims were to determine which day(s) during the holiday had highest motor vehicle fatality risk compared to non-holiday travel and to identify potential risk factors.Of 43,457 traffic fatalities studied, 15,292 (35%) occurred during the holiday, with Saturday being deadliest but Monday having highest odds of traffic fatality. Both sexes, all years, age < 65, drivers and passengers, rural and urban, and all regions in the United States were at increased risk during the holiday versus non-holiday periods. Motor vehicle crashes are a major concern in the United States, with more than 37,000 people killed in 2016, and an increase of nearly 2000 fatalities from 2015 to 2016 [Holidays are times of increased road travel. In 2003, the number of long-distance trips during holiday periods in the United States were higher than the average number for the rest of the year, and motor vehicle is one of the most popular modes of private recreational travel during the holidays in the United States . MemoriaThe primary aim of our study was to evaluate the risk of a fatal motor vehicle crash during the Memorial Day weekend and to determine which day(s) had the highest risk. Our second aim was to identify possible risk factors associated with odds of motor vehicle fatality during the Memorial Day weekend.The National Highway Traffic Safety Administration (NHTSA)’s Fatality Analysis Reporting System (FARS) is a national census of fatal traffic crashes in the United States . We usedHoliday fatalities were defined as traffic fatalities occurring during the Memorial Day holiday (Saturday through Monday). Non-holiday fatalities occurred during the corresponding 72 h precisely 1 week before and after the holiday. Seven factors were selected for analysis including day of week , year groups , age , sex , role , census region , and road location .p < 0.05.Descriptive analysis used Chi-square to compare holiday vs. non-holiday fatality counts and percentages. Because data for non-eventful driving are not available on a date-specific basis, we evaluated odds of holiday fatality using a binomial approximation. This method compares aggregated fatalities for the eventful exposure and eventful non-exposure groups by assuming that the number of non-fatalities are similarly large for Memorial Day weekends and non-holiday periods. An assumed non-eventful driving exposure of one million was used for all study days . In esseFrom 1981 to 2016, 43,557 fatalities occurred during the 3-day Memorial Day weekend  and six corresponding comparison days. The average number of deaths per day during the holiday was 142, compared to 132 the week before  and 128 the week after . For the holiday, Saturday had the highest number of fatalities  while Monday  had the lowest   , and individuals younger than 18 had 10% higher odds of traffic fatalities during the Memorial Day weekend . Females had 10% increased odds , and males had 9% increased odds of crash-related fatalities during Memorial Day weekend compared to control days . Odds of holiday traffic fatalities also differed by role with passengers 17% more likely  and drivers 5% more likely .Geographically, people in the Northeast experienced 16% increased odds of fatality during the Memorial Day weekend compared with control days . Regarding the crash location, the odds of the fatality on rural areas had 14% greater than the control days , and an 8% increase in urban areas .As expected, due to increased traffic volume and longer travel distances during holidays, holiday travel had higher odds of traffic fatality compared to non-holiday periods. Saturday was the deadliest day overall, as well as for holiday and control periods, although Memorial Day Monday had the highest odds of holiday fatality compared to control periods and the odds on Sunday were only marginally significant. Additionally, the increased travel during holidays can explain higher risk of holiday traffic deaths among adults and children . The higher odds of crash-related deaths during holiday periods might reflect children and their families travelling together for longer distances compared to typical work and school commutes. Public campaigns need to make efforts to remind drivers to expect increased congestions and delays during holidays.Nearly every category we analyzed had increased odds of a holiday traffic fatality, indicating that Memorial Day holiday travel patterns compared to typical travel are not isolated to certain demographic or geographic characteristics. The majority of motor vehicle crash fatalities were in urban areas during both holiday and control days likely due to nearly eighty percent of the U.S. population residing in urban areas , 14. HowA holiday, including Memorial Day weekend, is a chance to take off work pressure and enjoy time with friends and family members. Unfortunately, holidays are also associated with increased risky behaviors, such as drinking and other inappropriate driving behaviors . One traThe strength of our study was our use of a national census of all crash-related fatalities on public roadways from 1981 to 2016 with comprehensive information for all eligible fatal crashes. We also used an established method of comparing a case period with corresponding double control periods. This method can eliminate multiple unmeasured confounders, clarifies interpretation, and avoids misunderstanding.In our study, most of the factors with the increased risk during the holiday are the reflection of presumed increased exposure, such as increased holiday travel by distance, time, and increased traffic volume. Therefore, public campaigns are needed to improve road safety of all road users and focus on reminding travelers to expect delays and increased congestion.The main limitation of our study was data availability. The FARS dataset reports fatal crashes and therefore does not provide estimates of uneventful driving. National travel exposure data, such as estimated vehicle miles or time travelled, are annual estimates and not sensitive enough for date-specific comparisons. A traditional case–control study would have information for the health outcome , as well as exposure status . Using FARS, we were able to define case exposure status but were missing information for all controls. Therefore, a traditional multivariate regression analysis to calculate adjusted odds ratios was not possible.Table


The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1‐ARs. Here, using thermostabilized, purified, α1A‐ and α1B‐ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A‐ and α1B‐ARs. Similarly, in cell‐based assays expressing unmodified α1A‐ and α1B‐ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist‐stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A‐ and α1B‐ARs; however, this was shown to be caused by off‐target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.α ACadenylate cyclaseARadrenoceptorCaMcalmodulinCNScentral nervous systemCREcAMP response elementDDMn‐dodecyl β‐D‐maltopyranosideDMEMDulbecco's modified Eagle mediumGPCRG protein‐coupled receptorHEAT125I]‐2‐[[2‐(4‐hydroxy‐2‐iodophenyl)ethylamino]methyl]‐3,4‐dihydro‐2H‐naphthalen‐1‐one‐2‐[[2‐(4‐hydroxy‐2‐iodophenyl)ethylamino]methyl]‐3,4‐dihydro‐2H‐naphthalen‐1‐one (HEAT) on α1A/B/D‐AR expressing COS‐1 cells.3 assays demonstrated potentiation of agonist responses at α1A/B/D‐AR‐expressing cells by these benzodiazepines, suggestive of allosteric interaction. However, no further studies have been reported on the mechanism of benzodiazepine modulation of α1‐ARs, or the receptor binding site. We recently engineered thermostabilized mutants of α1A‐AR and α1B‐AR, which enabled binding epitope determination of orthosteric ligands with nuclear magnetic resonance (NMR) spectroscopy.1‐ARs so as to apply this information as a starting point for developing more selective modulators targeting this allosteric site. Instead we observed no evidence for the direct binding of several benzodiazepines to purified α1A‐AR and α1B‐AR using NMR and fluorescent ligand binding assays. Cell‐based binding and calcium signaling assays with wild‐type (WT) α1‐ARs also failed to detect any direct modulatory effects of diazepam on these receptors. While diazepam could positively modulate the stimulation of a cAMP response element (CRE) reporter through α1‐AR activation, this was found to be driven through the ability of diazepam to inhibit phosphodiesterases (PDEs), a known target of some benzodiazepines. This study highlights how purified GPCRs can be used to directly investigate allosteric modulator mechanisms that have been proposed from cell‐based assays, where off‐target actions are difficult to control for.Benzodiazepines are positive allosteric modulators (PAMs) of GABA22.1N‐hydroxyethyl‐nordiazepam was synthesized from nordiazepam according to the method of Archer et al.Nordiazepam and diazepam were synthesized from 2‐amino‐5‐chlorobenzophenone using the method of Sternbarch et al.2.21A‐AR variant A4 and α1B‐AR variant #15 were expressed in Escherichia coli and purified as described previously.1) was expressed and purified as described by Bumbak et al.® MyOne Streptavidin T1 paramagnetic beads  in 10 mL binding buffer , at 4°C for 1.5 hours. The receptor‐coated beads were washed in binding buffer, and then aliquoted into a KingFisher 96‐DeepWell™plate  at approximately 20 pmol of receptor per well. A Kingfisher 96 magnetic particle processor was then used to transfer the beads into plates containing QAPB and various competitors, which was incubated for 2 hours at 22°C with gentle mixing. The beads were then washed for 1 minute in binding buffer then transferred to 100 μL binding buffer in black Greiner nonbinding 96‐well plates. QAPB fluorescence was measured using 485/12 nm excitation and 520/10 nm emission filters, while mCherry fluorescence was measured using 544 nm excitation and 590/10 nm emission filters in an Omega POLARstar plate reader .α2.31A‐AR A4, or 5 μmol/L enNTS1, and 500 μmol/L test ligands with and without competition of 10 μmol/L prazosin in 500 μL of DDM buffer  in 5 mm NMR tubes. STD NMR was performed as described previously on a 700 MHz Bruker Avance IIIHD spectrometer using a cryogenically cooled triple resonance probeStock solutions of 100 mmol/L diazepam and lorazepam were prepared in deuterated DMSO. Stock solutions of 5 mmol/L prazosin were prepared in deuterated methanol. Saturation transfer difference (STD) NMR samples constituted 5 μmol/L α1H total correlation spectroscopy (TOCSY) and 1H correlated spectroscopy spectra acquired under the same solution conditions as the STD NMR experiments and in the presence of receptor. Spectra were typically acquired at 25°C with 10 ppm spectral widths and 2K data points in the direct dimension and 256 data points in the indirect dimension. TOCSY spectra were run with a spin lock time of 60 ms.Diazepam and lorazepam assignments were confirmed with two‐dimensional 2.41A‐AR and α1B‐AR as previously described.Saturation binding of QAPB in the absence or presence of 50 μmol/L diazepam was measured using COS‐7 cells stably expressing WT human α2+ mobilization assays were performed on nontransfected COS‐7 cells and cells stably expressing either α1A‐AR or α1B‐AR. Cells were seeded at 25 000 cells per well into 96‐well culture plates and allowed to grow overnight at 37°C, 5% CO2. Cells were washed twice with Ca2+ assay buffer  and then incubated in Ca2+ assay buffer containing 1 mmol/L Fluo‐4‐AM for 1 hour in the dark at 37°C and 5% CO2. After two washes with Ca2+ assay buffer and the addition of phenylephrine solutions (or co‐addition of phenylephrine and benzodiazepines) fluorescence was measured for 1.5 minute in a Flexstation plate reader  using an excitation wavelength of 485 nm and emission wavelength of 520 nm.Intracellular Ca2.51A‐ and α1B‐AR stably expressing COS‐7 cells, parental COS‐7 cells or parental HEK293T cells, with a β‐galactosidase expression plasmid under the control of the CRE promoter.2. Media was aspirated, and the cells frozen at −80°C for at least 24 hours before measurement of cellular β‐galactosidase expression using chlorophenol red‐β‐D‐galactopyranoside. Ligands were made up in 0.5% FBS DMEM media. Benidipine (10 μmol/L), bicuculline (30 μmol/L), IBMX (500 μmol/L), or rolipram (10 μmol/L) were co‐added with 0.1 μmol/L phenylephrine, or 10 μmol/L forskolin with and without diazepam or lorazepam (50 μmol/L). Inhibitors W‐7 hydrochloride (10 μmol/L), 2‐APB (40 μmol/L), (R)‐(+)‐Bay 8644 (4 μmol/L), and KN‐93 (20 μmol/L) were preincubated for 30 minute prior to addition of 50 μmol/L diazepam, and 10 μmol/L forskolin or 0.1 μmol/L phenylephrine for 6 hours. HEK293T cells transiently transfected with GPR68 and pCRE were stimulated with sodium bicarbonate‐free, low glucose DMEM, supplemented with 20 mmol/L HEPES and 0.5% FBS at pH 6.8, 7.0, 7.2, 7.4, and 7.8. The pH was adjusted at 37°C with NaOH. Cells were incubated for 6 hours at 37°C, in a room atmosphere incubator. Diazepam or lorazpam were made up in media at pH 7.2 or 7.8, co‐added with rolipram and stimulated for 6 hours in above conditions.cAMP response element (CRE) response assays were performed as previously described by transfecting WT α2.6Kd values calculated from separate measurements. Ca2+ mobilization data were performed in triplicate wells and normalized to the peak response elicited by 3 μmol/L ionomycin. CRE assay was conducted in triplicate, and data were normalized to the response elicited by the vehicle. Curves were fitted with three variable nonlinear regressions. For GPR68 assays, responses were normalized to CRE response at pH 6.8 (100%) and pH 7.8 (0%).Nuclear magnetic resonance (NMR) data were processed in Topspin 3.5 using squared cosine‐bells in both dimensions and zero‐filled once, prior to Fourier‐transformation. All other data was analyzed with Graphpad prism 7 . All error bars are standard deviation (SD) from three independent experiments. Competition binding assays were performed in duplicate wells. Curves were fit with one site nonlinear regression. For whole cell QAPB binding, the mean QAPB fluorescence intensity (MFI) of at lease 5000 cells was measured, and plotted against QAPB concentration. Values reported represent the mean and SD of at least three 31‐AR agonists in cell lines overexpressing α1‐ARs, with low micromolar potencies.1‐AR proteins has never been demonstrated. Thermostabilized GPCRs are receptors containing mutations that improve the protein stability upon solubilization and purification using detergents.1A‐AR A4 and α1B‐AR #15 were recently described and exhibit the stability required to probe the binding of benzodiazepines to the purified receptors.1‐AR, then they should influence the binding of orthosteric antagonists and/or agonists to α1‐AR. Biotinylated α1A‐AR A4 or α1B‐AR #15 was immobilized onto streptavidin‐coated paramagnetic beads and placed in a 10 nmol/L solution of fluorescent‐labeled BODIPY‐FL prazosin (QAPB), an approximately Kd concentration, with serial dilutions of validated competitors,diazepam or lorazepam, for 2 hours. The Kd of QAPB at α1A‐AR A4 and α1B‐AR #15 has been previously reported to be 11.6 and 8.5 nmol/L respectively.1‐AR agonist phenylephrine suggests that allosteric binding of diazepam influences the affinity of phenylephrine for the receptors. Thus, QAPB competition binding assays were performed at α1A‐AR A4 and α1B‐AR #15 where a sub‐IC50 concentration of phenylephrine (2.5 mmol/L) was included and the ability of increasing concentrations of diazepam to positively modulate QAPB displacement by phenylephrine was monitored. No cooperativity between diazepam and phenylephrine was observed at either receptor subtype. These data suggest that diazepam is either not binding to the thermostabilized α1‐ARs, has a very low affinity interaction, or is allosterically binding, but not able to modulate phenylephrine binding in this purified system.Previously the benzodiazepines lorazepam,diazepam, and midazolam were shown to behave as positive modulators of αKd > 1 μmol/L) and does not require labeled ligands or proteins.1A‐AR A4 and α1B‐AR #15.1A‐AR A4. STD NMR signals were observed for diazepam and lorazepam Saturation transfer difference (STD) NMR is a ligand‐observed experiment that is especially sensitive at monitoring ligands that bind weakly to proteins , which was not significantly different in the presence of 50 μmol/L diazepam (Kd = 20 ± 13 nmol/L)  and increasing concentrations of diazepam or lorazepam before measuring the mobilization of intracellular calcium 5 minute after treatment. Treatment with these benzodiazepines (0 to 50 μmol/L), had no effect in the absence . PLC catalyses the formation of diacylglycerol (DAG), which then activates phosphokinase C (PKC), and IP3 causing Ca2+ release from the endoplasmic reticulum via InsP3R Ca2+ channels.1‐AR activation also has a secondary effect of stimulating cAMP production via calmodulin (CaM)‐stimulated adenylate cyclase (AC).1‐AR CRE response. To assess the specificity of this action of diazepam, CRE assays were performed on human embryonic kidney (HEK) 293‐T cells, which endogenously express β‐ARs, treated with the β2‐AR agonist isoprenaline. Treatment with diazepam at 50 μmol/L significantly increased the CRE efficacy of isoprenaline at HEK293‐T cells  of benzodiazepines at their clinical target, GABAAR antagonist bicuculline had no effect on the positive modulation of phenylephrine or forskolin by diazepam ‐(+)‐Bay K 8644, and the Ca2+/CaM‐dependent protein kinase II inhibitor KN‐93, had no significant effect on the ability of diazepam to positively modulate the phenylephrine‐ or forskolin‐stimulated CRE responses (Figure Next, we sought to define the mechanism by which diazepam was modulating CRE activation by using inhibitors of various potential targets. Co‐addition of the GABAm Figure A,B. Volts Figure A,B. Simin Figure C,D. Thes1A‐AR expressing COS‐7 cells, IBMX and rolipram positively modulated the CRE response of phenylephrine to a similar level as diazepam (Figure 1‐AR CRE response by diazepam, and other benzodiazepines, is caused through inhibition of PDEs.Diazepam is an inhibitor of PDEs, especially PDE‐4,m Figure E; howevem Figure E. Similam Figure F. In facm Figure F, possibq, Gs, G12/13, and Gi/o proteins50 of pH 7.2 reflective of other reports1‐AR expressing COS‐7 cells (Figure Lorazepam was recently reported to be an allosteric modulator of the pH‐sensitive GPCR, GPR68.41‐ARs.1A‐AR, α1B‐AR, and α1D‐AR; however, direct binding of the benzodiazepines to α1‐ARs has never been measured. Here, purified, thermostabilized α1‐AR variants were used as a tool to probe whether diazepam and lorazepam positively modulate α1‐AR signaling via direct binding to the receptors. Interestingly, no evidence was obtained that diazepam or lorazepam could bind to either purified α1‐AR subtype using fluorescent ligand binding assay (Figure 1B‐AR and did not positively modulate phenylephrine‐induced intracellular Ca2+ release in cells expressing WT α1A‐AR or α1B‐AR (Figure The benzodiazepines diazepam, lorazepam and midazolam have been reported to be PAMs at the αy Figure . PossiblR Figure , also su1A‐AR and α1B‐AR expressing cells. The ability of diazepam to modulate the CRE response was independent of α1‐AR stimulation, which was shown using β‐AR agonists and cells that natively express β‐ARs,1A‐AR and α1B‐AR expressing cells, it enhanced the efficacy of phenylephrine only at α1B‐AR expressing cells. This likely reflects differences between how phenylephrine activates CRE at α1A‐AR compared to α1B‐AR.Using a CRE reporter assay, which is responsive to multiple GPCR‐stimulated signaling pathways to detect downstream effects, diazepam was found to positively modulate phenylephrine‐induced CRE stimulation in WT αA receptors is well understood. The substituent at C‐7 is of paramount importance; small electron‐withdrawing substituents at C‐7 generally impart high activity, whereas electron donors or large groups are inactive.A activity.AR (Table AR.The SAR governing benzodiazepine action at GABAAR Table , with th2‐ARs,2+ channels,2+/CaM‐dependent protein kinases50 of 9 μmol/L,3H‐rolipram at purified guinea pig PDE‐4,1‐ARs. This may be related to the fact that GPR68 can couple to Gs proteins to directly activate cAMP accumulation, and thus CRE,1‐ARs stimulate CRE indirectly through Gq.Benzodiazepine interactions have been reported against cholecystokinin receptors,1‐ARs to probe direct molecular interactions, allowing us to show that the modulation of α1‐AR activity by benzodiazepines in cell‐based assays is not a result of direct ligand binding. We further demonstrated that significant modulation of cellular CRE responses by diazepam most likely occurred through inhibition of PDE‐4. Given the widespread use of benzodiazepines therapeutically, this off‐target effect may contribute to their clinical actions and side effects and warrants further study. In contrast to this, lorazepam was validated as a direct‐acting, PAM of the pH sensitive receptor GPR68.In summary, this work shows the value of stabilized, purified α Click here for additional data file.


Allergic rhinitis (AR) is a global health problem that appears in all age groups and affects approximately 15–30% of people. Baicalin has been used for the treatment of various allergic diseases, including AR. However, the metabolic mechanisms of AR and baicalin against AR have not been systematically studied. Here, ovalbumin-sensitized AR rats were used as a model, and animal behaviour, histological analysis, enzyme-linked immunosorbent assay (ELISA) and metabolomics were used to elucidate the mechanism of baicalin for AR. The results indicated that baicalin has a protective effect on AR rats by inhibiting the release of immunoglobulin E (IgE), histamine, interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α). In addition, ovalbumin-induced AR included modulation of arachidonic acid, leukotriene A4 (LTA4), leukotriene B4 (LTB4), α-ketoglutaric acid, phosphatidylcholine PC (20 : 4/0 : 0), PC (16 : 0/0 : 0), citric acid, fumarate, malate, 3-methylhistidine, histamine and other amino acids that are involved in arachidonic acid, histidine metabolism, the TCA cycle and amino acid metabolism. Thus, AR could be alleviated or reversed by baicalin. It is aCurrent studies have predominantly concentrated on cytokines, chemokines such as interleukin-1 beta (IL-1β), tumour necrosis factor alpha (TNF-α) and interleukin-4 (IL-4), which are involved in the pathogenesis and process of allergic reactions during AR. However, the effective metabolites related to AR remain unclear ,9. It isScutellaria baicalensis Georgi, has been shown to have multiple pharmacological activities, including anti-allergic, anti-inflammatory and antioxidant effects ) and 554.2615[M − H] for leucine-enkephalin.Mass spectrometry was performed on a Waters Xevo G2 Q-TOF quadrupole accelerated time of flight mass spectrometer in positive and negative ion mode with an electrospray ionization (ESI) source. The desolvation gas was 800 l h2.5.2.The data processing was performed as previously described. Briefly, the raw MS spectra were analysed using MarkerLynx v. 4.1 . Then, the processed data list was exported and processed by orthogonal partial least-squares discriminant analysis (OPLS-DA) using the software package Simca v. 14.1 .2.6.p < 0.05 for this test.All values were expressed as the mean ± s.d. The significance of differences between the means of control, model and baicalin groups was compared through one-way ANOVA using the Statistical Package for Social Sciences program . The significance threshold was set at 3.3.1.−1.The nasal symptoms of rats were recorded for 15 min. The numbers of nasal sneezes and scratching significantly increased in the model group and significantly decreased in the baicalin group. The scores for the groups are shown in 3.2.Histological analysis was used to evaluate the pathological changes in the nasal mucosa of rats in each group. The nasal mucosa was arranged neatly and had no abnormality in the control group, while many eosinophilic granulocytes infiltrated the nasal mucosa, and blood stasis was clearly observed in the model group. The pathological changes were significantly alleviated by baicalin .Figure 3.3.−1 baicalin significantly decreased the levels of IgE, histamine, IL-1β, IL-4, IL-6 and TNF-α in the OVA-sensitized rats , HMDB (http://www.hmdb.ca) and KEGG (http://genome.jp/kegg) . The p-value was set to 0.05 when determining significantly different metabolites in this study. Next, 30 significantly different metabolites were selected for further study, and 21 metabolites were confirmed with available reference standards by matching their retention time and accurate elemental compositions.Reproducibility of the metabolomics approach was determined from six replicated analyses of the same quality control sample (QC) interspersed throughout the analysis. The relative standard deviation (RSD) of the peak area of all metabolites is below 20% , which demonstrates good stability and reproducibility . S-plot p < 0.05). The levels of linoleic acid, hippuric acid, lactic acid, LTB4, citric acid, hydroxyphenyllactic acid, fumarate, malate, 3-methylhistidine, arginine, tyrosine, histamine, N-acetylhistamine, asparagine, valine, LTA4 and creatinine were observed to be markedly increased in the model group (p < 0.05). In the baicalin-treated group, the apparent trend of these metabolite levels was the opposite of the model group.As shown in http://www.metaboanalyst.ca) was used to analyse metabolic pathways and showed that several pathways, such as arachidonic acid metabolism, citrate cycle, histidine metabolism, linoleic acid metabolism and other amino metabolism, were disordered in the AR model group or in the baicalin group , which can induce red blood cells and haemoglobin damage via oxidative mechanisms, is a critical component of polyunsaturated fatty acids  and can Furthermore, a large number of amino acids were significantly altered in AR. Our results suggested that rats sensitized by OVA may consume more energy in order to fight AR when compared to normal rats. Abnormal changes, such as fumarate, malate and α-ketoglutaric acid, belonging to the citric acid cycle, confirm this view. The citric acid cycle, as the linkage among sugar, lipids and amino acid metabolism, is the most important energy source for the body . In addiA similar study of AR treated by baicalin was recently published by another group using guinea pigs. The study focused on IgE, histamine and inflammatory cytokines, and the results suggested that baicalin may be a useful drug in the treatment of AR, which was the same as our results. Additionally, the index IL-8 was replaced with IL-4 in this study, and a randomized double-blind trial with AR showed that IL-4 is important in regulating IgE-mediated allergy ,29. IL-44.Our study indicated that baicalin has a protective effect on rats with AR by inhibiting the release of inflammatory mediators. Furthermore, metabolomics was applied to systematically study the AR induced by OVA and the mechanism of baicalin on AR. We conclude that baicalin is anti-AR not only because it suppressed the production of inflammatory mediators but also because it regulated arachidonic acid metabolism, the citric acid cycle, histidine metabolism, linoleic acid metabolism and other amino acid metabolism.


Human anatomical specimen museums are commonly used by medical, nursing, and paramedical students. Through dissection and prosection, the specimens housed in these museums allow students to appreciate the complex relationships of organs and structures in more detail than textbooks could provide. However, it may be difficult for students, particularly novices, to identify the various parts of these anatomical structures without additional explanations from a docent or supplemental illustrations. Recently, augmented reality (AR) has been used in many museum exhibits to display virtual objects in videos captured from the real world. This technology can significantly enhance the learning experience. In this study, three AR‐based support systems for tours in medical specimen museums were developed, and their usability and effectiveness for learning were examined. The first system was constructed using an AR marker. This system could display virtual label information for specimens by capturing AR markers using a tablet camera. Individual AR markers were required for all specimens, but their presence in and on the prosected specimens could also be obtrusive. The second system was developed to set the specimen image itself as an image marker, as most specimens were displayed in cross section. Visitors could then obtain the label information presented by AR without any markers intruding on the display or anatomical specimens. The third system was comprised of a head‐mounted display combined with a natural click interface. The system could provide visitors with an environment for the natural manipulation of virtual objects with future scalability. Medical museums exhibiting anatomical and pathological specimens first appeared in the 16th century Cole, . Many meAt the same time, numerous developments in information and communication technology have significantly influenced medical education. Medical educators have experimented with many recent developments, such as three‐dimensional (3D) printing  is a technology that enables the display of virtual objects in the real world by overlaying supplementary information on the real environment. In order to smoothly generate AR environments, the integration of spatial and temporal information in virtual and real environments is critical. Two methods are available for spatial information integration: the sensor‐based method and the image marker‐based method. The sensor‐based method identifies a position with high accuracy in the real environment using a sensor, such as a global positioning system (GPS) or an acceleration sensor. Augmented reality applications using handheld GPS devices have been previously utilized for field workers . This software allows users to engage with content, learn independently, and explore images of 3D anatomical structures. AR technology combined with 3DCG images can also be applied to anatomy education  is relatively smaller than some of the prominent museums cited earlier. The authors manage the museum and act as docents to present exhibitions to visitors . Medical students in the UYFM can visit the museum at any time during school hours, although only a small number of students actually do visit, probably because it is not included in the curriculum requirements for medical students, or because students are unaware of its presence. These students attend numerous lectures and engage in various activities, such as anatomy and pathology laboratory time . However, the number of total hours of basic biomedical science training has been decreasing in medical curricula, including that of gross anatomy teaching, along with an increase in case‐based learning relevant to health and disease in the form of either group interactive sessions or active learning  to optimize the AR techniques with which a real specimen in the museum is viewed to establish a new exhibition description and 2) to compare two AR systems alongside traditional description panels, with an additional learning experience evaluation. In each process, the present study prioritized the results from a subjective evaluation through questionnaire surveys in order to focus on user‐centered design. The first objective, which is the optimization of the AR techniques, included two‐step usability evaluations of AR tracking techniques  and display devices  via a pilot study with questionnaire surveys. The second objective, which is the comparison of two AR systems alongside traditional description panels, also involved a pilot study with a questionnaire survey on the perception of AR systems, with the addition of a learning experience evaluation. In the first evaluation of objective 1 , two systems with different AR tracking techniques were constructed: AR marker and object image marker systems (simply referred to as the image marker system). The AR marker system was developed using ARToolKit, version 2  and VisualStudio 2013 professional  on a desktop computer  and a tablet device . ARToolKit software is an open‐source library used to construct simple and low‐cost AR environments using AR markers. It enables a developer to easily implement processing steps, namely setting AR marker images, detecting captured images, and presenting information in the AR environment , a tour support system using a video see‐through head‐mounted display  was constructed. The HMD presented a display in front of both viewer eyes with a horizontal viewing angle of 31° and was connected to a laptop computer . Although the HMD device does not require users to hold the tablet toward each exhibit, it cannot perform touch‐screen operations. A more significant disadvantage is that it requires the operation of an additional computer keyboard connected to the HMD. A natural click interface technique  while using AR technology  aimed to compare the developed AR systems with the traditional methods for exhibition description from two viewpoints: effectiveness of learning  and a questionnaire survey on AR perception. A total of 84 participants studying anatomy from the first‐ and second‐year medical courses  were recruited. The participants were divided into three sample groups: control , tablet AR, and HMD AR. The first two groups each consisted of 32 participants, and the third group had 20 participants. The participants in the groups with the AR support experienced either of the two previous usability studies in order to be well accustomed with the device, and to simply compare the learning achievement in the three groups. The total time spent observing the specimens was measured from the video recording data using a portable device . Each participant freely visited eight pathological specimens arranged in three adjacent exhibition booths. A paper‐based pathology knowledge test consisting of eight questions with five choice answers was used to measure the learning effectiveness of the experiences (Appendix). Subsequently, the participants were asked to complete a questionnaire on their perception of the AR experience, their level of learning, and their satisfaction. The questionnaire was comprised of 10 statements for three themes  whether each system was useful , suggesting the superiority of the image marker system. Furthermore, the participants who responded “no” (68.3% (41/60) and 30.0% (18/60) for the AR marker and image marker, respectively) stated the following in their free comments: “The information of the AR marker system was not displayed when only the tablet’s camera captured each specimen image,” “The wait time for displaying information was somewhat frustrating in the image marker system,” and “Regardless of the system, holding the tablet became increasingly difficult (because of its weight) while observing each specimen.”Second, regarding ease (Q2), 31.7% (19 out of 60) of the participants responded “yes” for the AR marker system and 70.0% (42 out of 60) for the image marker system. The difference in the rate of positive responses was statistically significant . The large difference suggested the clear superiority of the image marker in appearance to the AR marker. The free comments of those who responded unfavorably were as follows: “AR markers appear to intrude on the viewing of the exhibits” or “I think AR markers disfigure the overall exhibition.”Third, in terms of the appearance question (Q3), all of the participants had favorable opinions of the image marker system, whereas only 30.0% (18/60) of the participants had positive opinions regarding the AR marker system. The difference was statistically significant . Several comments described some irritation with the wait time before display of AR information. Approximately one‐fourth of the participants (23.3% for tablet AR and 25% for HMD AR) disagreed, which could partially be due to this issue. Regarding device burden (Q3), the number of participants who agreed that the device was tiring to use was 68.3% (41/60) for the tablet AR and 25.0% (15/60) for HMD AR. The difference was statistically significant . Some of the comments on the positive or negative points in the free descriptions were are as follows: “I was comfortable with HMD because it was a completely hands‐free device, whereas I got tired of holding the tablet,” “I had some difficulty in viewing with the HMD display because of the small screen size and the low image resolution,” and “Continuing this visit for a longer period would induce dizziness with either of the device tools.”In the usefulness question (Q1) of the display device evaluation, all of the participants responded that both systems were useful. The AR‐based system was useful to users despite the different devices. Regarding ease (Q2), 76.7% (46/60) of the participants for the tablet AR and 75.0% (45/60) for the HMD AR responded that access to information was easy, respectively. No significant difference was found between them (χThese results suggest that (1) AR‐based systems are favorable to students regardless of the device and that (2) HMD avoids the physical inconvenience of carrying a tablet device, but may have disadvantages, such as poor visibility due to the small display size with low resolution.2 = 19.95, P < 0.001). The Wilcoxon multiple comparison showed that the total scores for both AR groups were significantly higher than that of the control group , whereas no significant difference was found in the scores between the two AR groups (P = 0.247). These results suggest that both AR groups could improve learning achievements regardless of the difference in the device. The mean values (±SD) of the total time spent were 3 minutes: 15 seconds (±1:23), 5 minutes: 01 second (±1:27), and 4 minutes: 25 seconds (±0:47) for the control, tablet AR, and HMD AR, respectively. A significant difference was found in the total time spent for the three groups . The Wilcoxon rank‐sum test with Bonferroni correction showed that the total time spent for both AR groups was significantly longer than that for the control . Use of the AR device was likely to extend the time spent observing the exhibition descriptions by increasing participant motivation and/or attention. In the tablet AR case, the time spent tended to be longer than that for the HMD AR group, but no significant difference was found (P = 0.518). These results suggest that, regardless of the device, AR information can facilitate learning activity among participants. The following free comments support this assumption: “I could follow the tour attentively (on the tablet AR),” “I regard the tools as fairly effective for learning (on the tablet AR),” “It was truly an inspiring experience with the new type of description. I would like to have more visits to other specimens as well (on the HMD AR),” and “It was not easy to get what the key points were, so I finished my tour immediately (on control).”The median placement accuracies of the total achievement test scores for the control, tablet AR, and HMD AR were 62.5%, 75.0%, and 68.8%, respectively Fig. . Signifi2 = 77.06, P < 0.001) . A significant difference was not observed in any pair of items in the comparisons between the tablet AR and the HMD AR. The overall results imply that AR support can facilitate learning activities by increasing students’ attention and/or motivation, and may thus lead to improved learning outcomes.The results from the questionnaire on perception showed that the scores for both AR groups were more than 4.0, whereas most of the scores for the control group were less than 4.0. A significant difference was found among the three groups  and perception. Although the number of samples and questions were small, the achieved score was significantly higher for the tablet AR and the HMD AR than for the traditional description panels. The results suggest that the AR‐based systems are not only effective tools for providing exhibition descriptions, but can also contribute to enhancing students’ motivation for learning. The evaluation of other items for the AR experience, level of learning, and satisfaction suggested an overall superiority of the AR systems to the traditional description panels. Meanwhile, no significant difference was found in the evaluation items between the tablet AR and the HMD AR, suggesting that AR information contents might provide a stronger effect than the differences in the device. According to the cognitive theory of multimedia learning or instructional design  additional labels for supplemental explanation such as anatomical 3DCG showing the 3D structural relation within the whole body or in relation to the vascular or the surrounding skeletomuscular system; (2) a smart system that can propose appropriate sets of AR information by estimating each visitor’s level or interest based on an analysis of their operations and that can even select optimum tour routes; and (3) hands‐on operations with gestures other than the natural click, such as sliding, pinching, and grasping. These approaches would facilitate active operating behavior for a more effective learning, thus providing an active learning framework.Another limitation is the financial matter of using mobile or wearable devices. A sufficient number of devices cannot be prepared at one time, especially in the case of school visits as a course curriculum, because all devices are prepared by curators. Application software that can be installed on each visitor’s mobile terminal would be a solution if strict conditions were secured for use exclusively during the tour without recording images. A low‐cost optical see‐through HMD with a high display resolution will be a better solution for avoiding symptoms of cybersickness in the future.This study developed AR‐based support systems to establish new learning modalities for tours in a medical specimen museum. To the best of the authors’ knowledge, this study is the first to report of a system that uses the specimen image as an image marker and employs a natural click interface for tours in a specimen museum. Based on the pilot survey results, the system was well optimized as a useful tool within the constraints of user‐centered design. The learning effectiveness of the AR‐based system was also confirmed. In conclusion, AR‐based systems not only can be effective tools for exhibition descriptions but can also contribute to enhancing students’ motivation for learning. The application of the AR technology within the anatomical museum may even provide a paradigm shift that could be expanded throughout the world, with AR‐based systems shaping the future of anatomical exhibitions. An HMD AR system with a natural click interface seems to be highly scalable in terms of not only the button arrangement but also other display contents. Further improvement of the system is expected to enhance the learning efficiency of anatomical education.ATSUSHI SUGIURA, Ph.D., is an assistant professor in the Interdisciplinary Graduate School at the University of Yamanashi, Chuo, Yamanashi, Japan. He teaches experimental medicine and physics and his research interests include augmented reality and computer and human vision.TOSHIHIRO KITAMA, Ph.D., is an associate professor at the Interdisciplinary Graduate School at the University of Yamanashi, Chuo, Yamanashi, Japan. He teaches physiology, anatomy, and experimental medicine, and his research interests include oculomotor and posture control in systems neuroscience. He is a member of the Society for Neuroscience (SfN).MASAHIRO TOYOURA, Ph.D., is an associate professor in the Interdisciplinary Graduate School, at the University of Yamanashi, Kofu, Yamanashi Japan. He teaches computer graphics and affective computing, and his research interests include augmented reality and computer and human vision. He is a member of the Association for Computing Machinery Special Interest Group on Computer Graphics and Interactive Techniques (ACM SIGRRAPH) and the Institute of Electrical and Electronics Engineers (IEEE) Computer Society.XIAOYANG MAO, Ph.D. is a professor at the Interdisciplinary Graduate School, University of Yamanashi, Kofu, Yamanashi Japan. She teaches computer visualization and discrete mathematics, and her research interests include texture synthesis and non‐photo‐realistic rendering and their applications to scientific visualization. She is a member of the Association for Computing Machinery Special Interest Group on Computer Graphics and Interactive Techniques (ACM SIGRRAPH) and the Institute of Electrical and Electronics Engineers (IEEE) Computer Society. Click here for additional data file.


Allergic rhinitis (AR) has been reported to be associated with chronic rhinosinusitis (CRS). The objective of this study was to investigate the effect of AR on nasal mucosa remodeling in CRS.n = 20), CRS with NP without AR , CRS without NP with AR , CRS without NP without AR , AR without CRS  and controls (n = 14). Eosinophil infiltration, mucus production, and collagen deposition were examined by hematoxylin and eosin, periodic acid schiff and masson’s trichrome staining, respectively. VEGF-A and microvessel density were detected by immunohistochemistry. The expression of remodeling markers, including TGF-β1, MMP-7, MMP-9 and TIMP-1 were measured by Western blot.Patients were enrolled and divided into the following groups: CRS with nasal polyps (NP) with allergic rhinitis (AR)(CRSwNPwAR; The expression of remodeling factors, including VEGF-A, CD31, CD34 and TIMP-1 were significantly increased in CRSwAR compared to CRSsAR. Goblet cell hyperplasia, as well as VEGF-A, CD31, CD34, and MMP-9 expression were significantly higher in CRSwNPwAR compared to CRSwNPsAR. However, the expression of collagen fibers, MMP-7 and TGF-β1 were significantly higher in CRSsNPwAR compared to CRSsNPsAR.AR could enhance the remodeling process in CRS. Moreover, AR had different effects on CRSwNP and CRSsNP. Chronic rhinosinusitis (CRS) is a chronic inflammation of the nasal cavity and paranasal sinus mucosa that affects 12.5% of the population worldwide . DiffereThe goals of treatment for CRS are to achieve a local disease control and reduce patients’ symptoms. Generally, conservative therapies, including topical application of steroids and nasal saline irrigations, are applied to patients with CRS to reduce underlying sinonasal inflammation. Surgical interventions, such as endoscopic sinus surgery, are reserved to improve sinus ventilation and mucociliary clearance for patients who fail maximal medical therapy. However, the current therapies for patients with CRS are unsatisfactory. It was estimated that up to 20% of CRS patients were not well-controlled by surgical interventions and required revision during the 5-year follow-up . Apart fCurrently, evidence suggested a relationship between inflammation and remodeling , 11. Lotn = 20), CRSwNP without AR , CRSsNP with AR , CRSsNP without AR , AR without CRS (n = 20) and patients with only anatomic variations without sinus diseases as controls (n = 14). This study was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No. WDRY2016-K002). Informed consent was obtained from every subject.This study was performed on patients who underwent endoscopic nasal surgery at the Department of Otorhinolaryngology-Head and Neck Surgery of the Renmin Hospital of Wuhan University  from January 2015 to December 2016. Patients were separated into the following six groups: CRSwNP with AR  scan in accordance with the recommended European diagnostic standard EPOS2012 . The diaNasal polyps tissues in patients with CRSwNPwAR or CRSwNPsAR were collected during the surgery. Uncinate process mucosa was collected from patients with CRSsNPwAR or CRSsNPsAR. The uncinate process mucosa of AR group was taken from patients underwent septoplasty. The inferior turbinate mucosa was collected from control patients who underwent septoplasty in the absence of sinus diseases and allergic disease. Tissues were collected at the time of surgery and divided into two portions. One was reserved in liquid nitrogen for protein extraction, while the other was fixed in 4% paraformaldehyde for 24 h prior to embedding in paraffin.The paraffin-embedded tissues were sectioned at a thickness of 5 µm for hematoxylin and eosin (HE), periodic acid Schiff (PAS) and Masson’s trichrome staining. All stains were performed in accordance with the manufacturer’s protocol. HE staining was performed to examine EOS infiltration in lamina propria and basement membrane thickness. Five randomly selected fields in each slice were analyzed with a constant mucosal length under a high-power field (× 400) by two separate investigators. PAS staining was used to evaluate mucus production in epithelia and glands. Positive-goblet cells were calculated in five randomly selected fields in each sample at 200× magnifications. Masson’s trichrome staining was performed to assess collagen deposition. Collagen volume fraction was quantified in five randomly selected fields at 200×magnifications using Image Pro Plus 6.0 .The tissues were cut into pieces and the total protein was extracted by RIPA lyses buffer . The total protein was quantitated by QuantiPro BCA assay kit  according to the manufacturer’s instructions. Protein samples (25 µg per lane) were separated by 10% SDS–PAGE gel and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 2 h at room temperature in TBST containing 5% skim milk. After 5 min washing in TBS, the membranes were subsequently incubated with monoclonal rabbit anti-human MMP7 primary antibody , monoclonal rabbit anti-human MMP9 primary antibody , monoclonal rabbit anti-human TIMP1 primary antibody , polyclonal rabbit anti-human TGF-β1 primary antibody  or monoclonal rabbit anti-human β-actin primary antibody  at 4 °C overnight. Following extensive washing in TBST, the membranes were incubated with polyclonal goat anti-rabbit secondary antibody conjugated with horseradish peroxidase  for 1 h at room temperature. Immunoreactive bands were detected by the electrochemiluminescence Western blotting detection system .Immunohistochemistry (IHC) procedure was performed using the streptavidin biotin complex (SABC) kit . Briefly, after deparaffinization and rehydration, the tissue sections were microwaved for 10 min in citrate buffer for antigen retrieval and then incubated separately with primary rabbit polyclonal antibody against CD31  and primary rabbit polyclonal antibody against CD34  overnight at 4 °C to stain target protein expression. Following 20-min incubation with secondary anti-rabbit antibody, 3,3-diaminobenzidine (DAB) was applied for visualizing immunoreactivity. Five randomly selected fields in each slice were quantified and the integrated optical density (IOD) immunoreactivity was calculated as the relative products of the stained area using Image Pro Plus 6.0 .p < 0.05 was regarded as statistically significant.Experimental results were expressed as mean ± SD. Data analysis was conducted using SPSS software . Clinical characteristics were compared among groups using Chi square test. One-way ANOVA with Tukey analysis was performed to compare variabilities among groups. A level of p > 0.05). The LK endoscopy scores were significantly higher in the CRSwNPwAR group compared to the CRSsNPwAR group.The clinical characteristics summarized for all patients are shown in Table The HE staining showed that the number of EOS in CRSwAR group was significantly higher compared to the CRSsAR group . Moreover, the number of EOS in CRSwNPwAR group was significantly higher compared to the CRSwNPsAR group.And, the number of EOS in the CRSsNPwAR group was significantly higher compared to the CRSsNPsAR group .To assess the effect of AR on nasal mucosa remodeling in CRS, VEGF-A was detected by IHC. The results revealed that the VEGF-A expression was significantly increased in CRSwAR group compared to CRSsAR group . The expression of VEGF-A in CRSwNPwAR group was higher compared to CRSwNPsAR group . However, no significant difference was observed between CRSsNPsAR group and CRSsNPwAR group  and CRS are all characterized by both inflammation and remodeling. Researchers found a close relationship between inflammation and remodeling , 21. TraVEGF is widely recognized as a major proangiogenic factor involved in the process of neovascularization and vascular leakage through VEGF receptor-mediated pathways in tissue remodeling . PreviouMMPs is a family of proteolytic enzymes that implicated tissue remodeling, proliferation and migration by breaking down ECM components, facilitating epithelial cell migration and modulating the state of cell–matrix and cell–cell interactions . MMPs anTGF-β, a multifunctional cytokine that can regulate cell proliferation, differentiation, and migration, is associated with a variety of inflammatory disorders . New eviA limitation of this study is that we did not investigate the correlation between severity of AR and remodeling degree in CRS. Further studies on relationship between severity of AR and remodeling in CRS are needed.In conclusion, our results suggest that AR could enhance the remodeling process in CRS. Moreover, we found AR had different effects on CRSwNP and CRSsNP. For AR enhanced goblet cell hyperplasia, as well as VEGF-A, microvessel density, and MMP-9 expression in CRSwNP and promoted the expression of collagen fibers, TGF-β1 and MMP-7 in CRSsNP.


Androgen receptor splice variant 7 (AR-V7) results in a truncated receptor, which leads to ligand-independent constitutive activation that is not inhibited by anti-androgen therapies, including abiraterone or enzalutamide. Given that previous reports suggested that circulating tumor cell (CTC) AR-V7 detection is a poor prognostic indicator for the clinical efficacy of secondary hormone therapies, we conducted a prospective multicenter validation study.ClinicalTrials.gov identifier: NCT02269982) is a multicenter, prospective-blinded study of men with high-risk mCRPC starting abiraterone acetate or enzalutamide treatment. The primary objective was to validate the prognostic significance of baseline CTC AR-V7 on the basis of radiographic or clinical progression free-survival (PFS) by using the Johns Hopkins University modified-AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay. Overall survival (OS) and prostate-specific antigen responses were secondary end points.PROPHECY  and OS  after adjusting for CTC number and clinical prognostic factors. Men with AR-V7–positive mCRPC had fewer confirmed prostate-specific antigen responses (0% to 11%) or soft tissue responses (0% to 6%). The observed percentage agreement between the two AR-V7 assays was 82%.We enrolled 118 men with mCRPC who were starting abiraterone or enzalutamide treatment. AR-V7 detection by both the Johns Hopkins and Epic AR-V7 assays was independently associated with shorter PFS (hazard ratio, 1.9 [95% CI, 1.1 to 3.3; Detection of AR-V7 in CTCs by two blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, and such men with mCRPC should be offered alternative treatments. Moreover, cross-resistance is common,5 and clinical features are unable to predict cross-resistance. Thus, predictive biomarkers are urgently needed to optimize treatment selection.Men with metastatic castration-resistant prostate cancer (mCRPC) have improved survival when treated with the androgen receptor (AR) inhibitors enzalutamide or abiraterone.8 In addition, constitutive activation through the expression of AR splice isoforms that lack the androgen ligand-binding domain contributes to resistance.12 Detection of the splice isoform AR-V7 in circulating tumor cells (CTCs) has been strongly associated with abiraterone or enzalutamide resistance15 but was compatible with responsiveness to taxane chemotherapy.16 These data suggest that Androgen receptor splice variant 7 (AR-V7) assays may provide predictive utility to guide treatment decisions; however, prospective, multicenter validation is needed. We report the results of PROPHECY , an independent, multicenter, prospective-blinded validation study of two CTC AR-V7 assays in predicting PFS and OS with abiraterone or enzalutamide in men with mCRPC.In many men with mCRPC, the AR pathway remains active despite testicular androgen suppression through cancer-specific AR upregulation, mutation, and paracrine/autocrine androgen synthesis.18  who unblinded the data after the database was locked.This prospective, multicenter study evaluated the ability of baseline (pretreatment) AR-V7 status in CTCs to predict treatment outcomes with abiraterone or enzalutamide. All the authors vouch for the completeness and integrity of the data and for the fidelity of the study to the clinical protocol (Data Supplement). Peripheral blood samples for analysis of CTCs were obtained from eligible patients at prespecified time points: baseline and at the time of clinical, radiographic, or biochemical progression. CELLSEARCH  CTC enumeration was performed at these time points on all patients and processed in a College of American Pathologists/Clinical Laboratory Improvement Amendments–approved central laboratory.22 The Johns Hopkins University  modified-AdnaTest CTC AR-V7 mRNA assay was performed as previously described using validated methods.25 Established standard operating procedures for sample collection, overnight shipping, processing, and analysis were followed by study sites and the central laboratory at JHU.24 The Data Supplement details additional methods, including CTC heterogeneity criteria using the Shannon index.22CTCs were analyzed in two central laboratories, each blinded to the results of the other. CTC identification by the Epic Sciences  CTC nuclear-specific AR-V7 protein assay and CTC heterogeneity evaluations were performed as described previously.26 Clinical progression was defined by death, pain, or other symptomatic progression; initiation of new systemic therapy; or a skeletal-related event. Secondary clinical end points included confirmed 50% or greater prostate-specific antigen (PSA) declines, radiographic response per RECIST version 1.1,27 and OS. PSA declines were confirmed with a subsequent PSA value 2 or more weeks later.The primary end point was PFS, defined from date of registration to clinical/radiographic progression or death, whichever occurred first. Radiographic progression was assessed using Prostate Cancer Working Group 2 soft tissue and bone scan criteria.v 6 months. The following assumptions were made: binary AR-V7 status (positive and negative), a prevalence of AR-V7 positivity of 30%, and that PFS would follow an exponential distribution. In addition, the log-rank test had 80% power to detect an HR of 2.68 if the true prevalence of AR-V7 positivity was 10% or greater.The primary objective was to validate that AR-V7–negative patients have prolonged PFS with abiraterone or enzalutamide compared with AR-V7–positive patients at the trial level. OS and response rates (PSA and radiographic) were secondary clinical outcomes. The null hypothesis was that the hazard ratio (HR) of PFS in the two groups is 1.0 versus the alternative hypothesis that the HR of AR-V7–positive to AR-V7–negative patients is 2.0. With 90 projected PFS events, the log-rank test has 85% power to detect an HR of 2.0, equivalent to an improvement in the median PFS of 3 28 prognostic factors (risk score), including PSA level, alkaline phosphatase, lactate dehydrogenase, opioid analgesic use, Eastern Cooperative Oncology Group performance status, albumin, hemoglobin, and metastatic site . The Kaplan-Meier method was used to estimate median PFS and OS distributions by AR-V7 status. As a secondary biomarker analysis, logistic regression and proportional hazards models were used to test for the prognostic significance of a high CTC heterogeneity score (defined as a Shannon index ≥ 1.5) in predicting a 50% or greater PSA decline, objective response, PFS, and OS.Patients with no evaluable CTCs were considered AR-V7 negative, and all patients with sufficient blood collection were analyzed regardless of their evaluable CTCs. In secondary analyses, the proportional hazards model was used for assessing the prognostic value of AR-V7 status for PFS and OS adjusted for Halabi et alBetween May 2015 and January 2017, we enrolled 118 men with high-risk mCRPC from five academic medical centers (Data Supplement). Baseline characteristics of the cohort are listed in Of the enrolled men, 55 were treated with abiraterone, 58 were treated with enzalutamide, and five received both therapies concurrently. With no anticipated or observed differences in outcome (PFS or OS) between therapies, results were combined for the primary analysis (Data Supplement). The median follow-up time among surviving patients was 19.6 months, with 102 PFS events and 53 deaths as of the cutoff date of April 9, 2018, when the clinical database was locked for the primary end point. Median PFS was 5.8 months , and median OS was 20.3 months  for the overall cohort, which reflects the high-risk features of this population.At baseline, 28 men (24%) were AR-V7 positive, 88 (75%) were AR-V7 negative, and two (1%) were unevaluable by the JHU mRNA assay. By comparison, 11 men (9%) were AR-V7 positive, whereas 96 (82%) were AR-V7 negative, and 11 (9%) were unevaluable by the Epic protein-based assay (Data Supplement). The percentage agreement between the two CTC AR-V7 assays was 82% (86 of 105). Most discordant results (17 of 19) were JHU AR-V7 positive but Epic AR-V7 negative (Data Supplement). AR-V7 detection at baseline differed by assay and according to disease burden, clinical prognostic factors, and prior therapy (Data Supplement).v 6.9 months, respectively . For the Epic AR-V7 protein assay, the median PFS for AR-V7–positive versus AR-V7–negative patients was 3.1 v 6.1 months, respectively . OS differed widely according to AR-V7 status. For the JHU AR-V7 mRNA assay, median OS for AR-V7–positive versus AR-V7–negative patients was 10.8 v 27.2 months, respectively . For the Epic AR-V7 protein assay, the median OS for AR-V7–positive versus AR-V7–negative patients was 8.4 v 25.5 months, respectively . The primary end point of PFS was significantly different in AR-V7–positive men with mCRPC compared with AR-V7–negative men for both AR-V7 assays. For the JHU AR-V7 assay, the median PFS for AR-V7–positive versus AR-V7–negative patients was 3.1 28 and CTC AR-V7 by the JHU assay was significantly associated with worse PFS . The Epic AR-V7 assay also was associated with worse PFS . Halabi risk score and CELLSEARCH CTC enumeration were not associated with PFS after adjustment for AR-V7 status.In a multivariable analysis of baseline AR-V7 status, adjustment for baseline CTC enumeration (CELLSEARCH), Halabi prognostic risk score,CTC AR-V7 detection by both assays was independently associated with worse OS in multivariable analysis. The HRs for death for AR-V7 positivity were 4.2  and 3.5  for the JHU and Epic assays, respectively (Data Supplement). Halabi risk score and CELLSEARCH CTC enumeration were not associated with OS after adjustment for AR-V7 status, but risk score was associated with PFS and OS in JHU AR-V7–negative patients (Data Supplement). OS was 12 or fewer months after abiraterone or enzalutamide initiation for 63% of Epic AR-V7–positive men (seven of 11), 54% of JHU AR-V7–positive men (15 of 28), 47% of JHU AR-V7–positive and Epic AR-V7–negative men (eight of 17), and 14% of AR-V7–negative men by either test (11 of 77).For the key secondary outcomes of best overall confirmed PSA decline or soft tissue responses with abiraterone or enzalutamide treatment, no Epic AR-V7–positive patients had a confirmed PSA or RECIST response. Eleven percent of JHU AR-V7–positive patients had a confirmed PSA response, and 6% (three of 51) had a RECIST response. By comparison, there were 26% to 28% with confirmed PSA responses and 21% to 25% with soft tissue responses in AR-V7–negative patients by either assay . PSA dec22 A high Shannon index was associated with poor OS  and poor PFS  in univariable analysis and a lower proportion of confirmed PSA responses . Epic AR-V7–positive patients were more likely to have high CTC heterogeneity previously (Shannon index ≥ 1.5), which indicates a higher diversity of cellular phenotypes. Sixty-four percent of AR-V7–positive men (seven of 11) had high heterogeneity versus only 14% (13 of 96) of AR-V–negative men. AR-V7 positivity by the JHU assay was independently associated with PFS  and OS  after adjusting for CTC number and Shannon index, whereas the Shannon index and CTC number were not significantly associated with PFS or OS (P not significant). Epic AR-V7 positivity also was associated with PFS  and OS , whereas CTC number and Shannon index were not associated with either PFS or OS (P not significant).Finally, we examined the relationship of AR-V7 heterogeneity within patients and over time and the relationship of AR-V7 to high CTC phenotypic heterogeneity denoted by an elevated Shannon index.Finally, although the majority of CTCs in men with mCRPC were AR-V7 negative, even in AR-V7–positive patients, the proportion of AR-V7–positive cells ranged from 1% to 100%  of 28 JHU AR-V7–positive men having PFS times of more than 12 months and only one (9%) of 11 Epic AR-V7–positive men having a PFS time of more than 6 months. Thus, knowledge of the AR-V7 status using one of these blood-based assays, in conjunction with standard clinical prognostic measures, may predict the probability of benefit from abiraterone or enzalutamide.31 glucocorticoid receptor activation,32 AR gain or ligand binding domain mutations,35 alternative AR variants and genomic structural rearrangements,38 AR enhancer amplification,39 and additional compensatory oncogenic pathways.33 PSA declines with abiraterone or enzalutamide are associated with improved PFS and OS, which supports PSA monitoring for AR-V7–negative men with mCRPC.40 AR-V7 likely explains up to 25% of AR therapy resistance, which implies that most treatment resistance mechanisms remain unidentified. Although the JHU assay resulted in more AR-V7–positive patients versus the Epic assay (24% v 9%), these differences may relate to CTC detection differences between assays and the greater sensitivity needs of nuclear AR-V7 protein detection. Despite this, men who tested positive by the JHU AR-V7 assay were confirmed to have poor outcomes and a low probability of response to abiraterone or enzalutamide. Trade-offs between assay sensitivity and specificity for the prediction of response to abiraterone or enzalutamide are clearly present, with the Epic assay providing no false-positive results and the JHU assay detecting twice as many AR-V7–positive men but resulting in 6% to 11% of AR-V7–positive men with confirmed PSA/radiographic responses. Critical to the development of a precision medicine algorithm for men with mCRPC will be the standardization and clinical validation of assays that capture novel mechanisms in a timely manner for consideration in treatment decisions. Our data support AR-V7 as one such important prognostic biomarker for mCRPC.Our findings suggest that although a positive AR-V7 test is associated strongly with hormonal resistance, high-risk AR-V7–negative men may still not respond to AR inhibition, despite a greater probability of response. Alternative resistance mechanisms independent of AR-V7 include lineage plasticity and AR indifference,22 demonstrated a direct correlation between heterogeneity and AR-V7 positivity, which suggests greater tumor cell entropy and inherent resistance to AR targeting in these patients. Of note, we find that the negative prognostic and predictive significance of AR-V7 testing using either assay was independent of the number of CTCs,20 CTC heterogeneity, and other clinical prognostic measures of disease burden,42 which support the hypothesis that AR-V7 may be causally related to these poor outcomes and treatment resistance. These results, coupled with preclinical mechanistic studies demonstrating ligand-independent activation of AR-V7 in promoting the AR transcriptional program and treatment resistance12 and the increased detection of AR-V7 in some progressing patients on AR inhibiting therapies,9 support the concept that AR-V7 is associated with both phenotypic heterogeneity and AR therapy resistance. Trials are still needed to address whether AR-V7 is a driver of disease resistance through therapeutic targeting of AR N-terminal or DNA-binding domains. Our data suggest that AR-V7 is highly associated with rapid resistance to hormonal therapy and disease heterogeneity and is enriched at progression during treatment with AR inhibitors even after adjusting for disease burden and CTC enumeration.An objective measure of cellular heterogeneity using the Epic platform (Shannon index)16 Testing patients with multiple poor-risk prognostic features similar to those included in our study could therefore inform the decision to proceed with hormonal therapy or docetaxel chemotherapy. AR-V7–positive men with mCRPC still have a reasonable probability of response and clinical benefit with chemotherapy.15 Hence, the current results will inform clinical practice given the confirmed low probability of benefit with current AR inhibitors in AR-V7–positive men, particularly in those previously exposed to potent AR inhibitors. A recent study that evaluated the real-world clinical utility of AR-V7 testing suggested a therapeutic benefit using a biomarker-informed (rather than a biomarker-agnostic) approach in the management of mCRPC.25 A second limitation is the inclusion of only poor-risk men who were more likely to have CTCs and informative results; men with no CTCs and a more favorable prognosis, particularly in the first-line setting, will likely test negative for AR-V7 and may not benefit from AR-V7 testing.24 Finally, although our sample size was sufficient for independent multivariable prognostic validation of AR-V7, there were only 11 and 28 men who tested positive for AR-V7 by the Epic and JHU assays, respectively, which limits power for broader multivariable analyses. Larger controlled studies that more comprehensively assess CRPC genotypes, phenotypes, and AR splice variants are needed to confirm the predictive value of AR-V7 in the context of the host and CRPC genomic factors.One limitation of our study is the lack of testing with alternative treatment strategies in AR-V7–positive men with mCRPC, such as docetaxel chemotherapy. However, prior work suggests that AR-V7 positivity by either assay is adversely prognostic in men with mCRPC but is associated with better outcomes and response to taxane chemotherapy compared with poor outcomes with AR inhibitors in this population.In conclusion, we have prospectively demonstrated that AR-V7 is a strong predictor of clinical outcomes in men with mCRPC treated with abiraterone or enzalutamide. In patients with multiple clinical indicators of poor prognosis, we can identify a significant subset of patients with detectable AR-V7 by two independent assays. Knowledge of AR-V7 status may optimize treatment selection beyond clinical measures of prognosis and disease burden. The PROPHECY study represents a multicenter effort that provides prospective, blinded clinical validation around such an approach and suggests that both the JHU CTC AR-V7 mRNA assay and the Epic CTC nuclear-specific AR-V7 protein assay provide clinical utility around the anticipated outcomes with therapy.


Upbeat, an augmented reality (AR) dance game designed to improve rehabilitation therapies in upper limb amputees. In Upbeat, the patient is instructed to follow a virtual dance instructor, performing choreographed dance movements containing hand gestures involved in upper limb rehabilitation therapy. The patient's position is then tracked using a Microsoft Kinect sensor while the hand gestures are analyzed using EMG data collected from a Myo Armband. Additionally, a gamified score is calculated based on how many gestures and movements were correctly performed. Upon completion of the game, a diagnostic summary of the results is shown in the form of a graph summarizing the collected EMG data, as well as with a video displaying an augmented visualization of the patient's upper arm muscle activity during gameplay. By gamifying the rehabilitation process, Upbeat has the potential to improve therapy on upper limb amputees by enabling the start of rehabilitation immediately after trauma, providing personalized feedback which professionals can utilize to accurately assess patient's progress, and increasing patient excitement, therefore increasing patient willingness to complete rehabilitation. This paper is concerned with the description and evaluation of our prototypic implementation of Upbeat that will serve as the basis for conducting clinical studies to evaluate its impact on rehabilitation.Unsuccessful rehabilitation therapy is a widespread issue amongst modern day amputees. Of the estimated 10 million amputees worldwide, 3 million of whom are upper limb amputees, a large majority are discontent and experience rejection with their current prosthesis during activities of daily living (ADL). Here we introduce  Limb loss is a recurrent problem all across the world. Every year, an estimated 185,000 people undergo upper limb amputations , 3. EffoA common cause of prosthetic rejection is unsuccessful rehabilitation therapy, in which the amputee is unable to develop the sufficient skills needed to successfully manage their prosthesis during ADLs . Some ofAugmented and virtual reality (AR/VR) has the potential to ameliorate the rehabilitation process. By using a virtual arm instead of waiting for a prosthetic, patients can start rehabilitation immediately after trauma, consequently reducing the acuteness of muscle atrophy . An examAnother issue with current rehabilitation in upper limb amputees is the lack of a comprehensive objective assessment of the patient's progress. Commonly used methods to evaluate patient progress include the Box and Block Test (BBT) , the SouAmong the solutions to tackle the lack of comprehensive assessment mentioned above stands the protocol developed by Chadwell et al. , which cUpbeat's implementation, integration of motion tracking and electromyography (EMG) sensors within an AR system provides quantitative data physicians can use for objective assessment of patient progress. The flexibility of such a system also allows the therapy to be personalized to each patient's unique needs.A year later, Hunt et al. developed the Prosthetic Hand Assessment Measure (PHAM), an alternative method to quantitatively assess performance in a range of manipulation tasks associated with object manipulation , Play, and Feedback. Navigation throughout the application can be done with either mouse inputs or with gestures detected from the Myo Armband . The PlaThe hand gesture prompt is an image icon on the bottom right corner of the screen that informs the patient of which hand gesture to perform at the given moment. Hand gestures are tracked with the Myo Armband, and if the correct gesture is performed in the expected time frame, the image is replaced with a green success symbol to tell the patient that correct gesture is performed. In the final Feedback scene , the patUpbeat was developed with the 3D game engine Unity, through which all the sensors and software used  were inMirracle , an AR aUpbeat was choreographed and recorded with the Kinect. The motion-capture data were then used to animate the in-game virtual dance instructor which later guides the patient through the same movements. The choreography consists of a set of dance movements embedded with hand gestures inspired by the PHAM model. The dance choreography was designed to include full range of motion in the upper body, as well as regular, repetitive movements. These two factors (movements with various degrees of freedom and repetitions) have proven to be beneficial in state-of-the-art rehabilitation therapies [The integrated dance used in herapies .Each dance move includes one of the four hand gestures   see . The gesDLs) see . These gBlender.Org, 2018, https://www.blender.org. Accessed 29 Nov 2018.), in the form of a rig, an animated 3D skeleton, which was later used to animate the dance instructor virtual model for the game . The motion data from the NI mate rigging were matched to the model's body , which was a clearly visible location for the patient. In addition, speakers were connected to the computer and used to play the music for the dance game.Myo Connect. In the calibration, the user is prompted to perform each of the five recognizable gestures  a sequence of times. The process takes approximately 3-4 minutes overall, and it has to be done every time a new user interacts with the system.Before the gameplay begins, each user undergoes a calibration protocol for the Myo Armband to ensure correct hand gesture detection. Calibration is done through the Myo Armband's proprietary interface, The recording of the patient's performance during the game is displayed in real time on the game's background, simulating the effect of a mirror. This setup is the most appropriate in order for the patients to clearly see the virtual dance instructor, as well as their own mirrored reflection (from the head to slightly above their knees), such that they could perform the dance movements with as much visual observance as possible.Mirracle AR mirror system [Performance consisted in three key elements: gesture completion, muscle activity, and muscle activation. The Myo Armband was integrated into the application to track EMG activity and detect the hand gestures. The gesture completion is evaluated using a score based on the number of successfully completed gestures. The patient's muscle activity is then shown using an EMG graph, while a color-coded visualization of muscle activation is produced with the r system .The postgame feedback is designed to deliver a comprehensive evaluation of the user's performance, following methods that have proven to be effective for rehabilitation , 12. TheThe scoring system is based on the timing and accuracy of gesture completion. Each dance step shown by the virtual dance instructor contains one hand gesture that needs to be matched by the patient. Whether or not the patient successfully completes the gesture is tracked by the Myo Armband, which analyzes muscle activity in order to classify the movement into a recognized gesture. If the performed gesture both matches the one shown by the dance instructor and is performed within a certain time frame, it is deemed as the correct movement and adds 100 points to the player's total score. The time frame for each specific hand gesture and dance movement varies based on the choreography and music, but typically lasts between 6 and 12 seconds. The total game score is then calculated based on how many hand gestures are accurately performed by the player throughout the game, with points awarded for each successfully completed gesture.The Myo Armband contains an array of 8 bipolar surface electrodes that measure the EMG activity from the user. The raw data are then streamed wirelessly through the Myo Data Capture application at a frequency of 200 Mhz to populate a.csv file  that is later used to produce a graph of the patient's EMG activity.This graph is displayed upon completion of the game in the Feedback scene. Each color in the graph represents data collected by each individual sensor, and the overall analysis can be used by the practitioner to visually analyze the muscle activity patterns as an indication of the patient's progress through the rehabilitation process.Mirracle application records a video of the patient performing the movements during the gameplay. It uses this video in combination with the Kinect depth sensor data to output the same video with an augmentation of the musculoskeletal system of the upper arm overlaid on top of the patient's right arm. The augmented muscles are color-coded  in real time to indicate the muscles being used.In our system, the Upbeat environment is important in order to evaluate whether or not the Myo Armband functions properly in a Unity environment.The system was evaluated on three subjects, with 10 trials of the gameplay performed by each subject. In each trial, we measured the system's ability to correctly classify each specific gesture. Even though the classification is performed using the built-in Myo Armband software, measuring the classification accuracy within the ot) as the time taken for each hand gesture to be detected by the system from the moment it appears on the screen. Operating time can be broken down into detection and reaction times (dt) is defined as the time it takes for each specific gesture to be recognized by the system, while reaction time (rt) is defined as the time it takes the subject to perform a hand gesture from the moment it appears on the screen . Detecte screen .(1)ot=rtThe results for each hand gesture class are shown in Upbeat would involve faster dance movements expected to be performed within a shorter time period, it is crucial for the system to be able to detect different gestures quickly and accurately, in order to accommodate for the different levels of proficiency for each patient as their rehabilitation therapy progresses. To assess the system's ability to accommodate faster dance movements, we used the detection time data to compute the percentage of hand gestures that could be efficiently detected within a time interval of no more than four seconds .In order to make the system appropriate for rehabilitation, the next step is to implement a more complicated set of hand gestures better reflecting those used in PHAM . This prUpbeat in comparison to that of a control group following traditional rehabilitation therapy. As the system is intended to improve rehabilitation in upper limb amputees, it is important to understand how a system like Upbeat is received by its target group. Furthermore, while the qualitative and analytical aspects of the system already have strong support, both from this study as well as related studies, a more subjective assessment on how enjoyable an application like Upbeat will be is a future target of study.Most importantly, a clinical study with a group of upper limb amputees shall be conducted in order to evaluate their progress when using Upbeat converts the proven success of PHAM rehabilitation therapy and transforms it into a fun and enjoyable gamified experience for rehabilitation. As such, the gamified aspect of Upbeat has the potential to improve the rehabilitation process by increasing user's excitement. Portability of the system allows for rehabilitation to begin immediately after trauma, rather than waiting for prosthetics to be made or for medical-guided therapies to be concretely established. Upbeat further facilitates the important element of personalized feedback which can prove to be essential for the amputee to understand their progress, as well as giving doctors the ability to simultaneously track the progress without being overbearing on the rehabilitative process. Upbeat is presented as a prototype for gamified AR rehabilitation therapy and, in future work, will be used to conduct a clinical trial to evaluate its efficacy in achieving the envisioned goals.