Background: In persons with Parkinson’s disease (pwPD) any additional somatosensory or distractor interference can influence the posture. When deprivation of vision and dual-task are associated, the effect on biomechanical performance is less consistent. The aim of this study was to evaluate the role of the visual deprivation and a cognitive task on the static balance in earlier stage PD subjects. Methods: Fifteen off-medication state pwPD (9 women and 6 men), 67.7 ± 7.3 years old, diagnosed PD since 5.4 ± 3.4 years, only Hoehn and Yahr state 2 and fifteen young control adults (7 women and 8 men) aged 24.9 ± 4.9 years, performed semi-tandem task under four randomized experimental conditions: eyes opened single-task, eyes closed single-task, eyes opened dual-task and eyes closed dual-task. The center of pressure (COP) was measured using a force plate and electromyography signals (EMG) of the ankle/hip muscles were recorded. Traditional parameters, including COP pathway length, ellipse area, mediolateral/anteroposterior root-mean-square and non-linear measurements were computed. The effect of vision privation, cognitive task, and vision X cognitive was investigated by a 2 (eyes opened/eyes closed) × 2  repeated-measures ANOVA after application of a Bonferroni pairwise correction for multiple comparisons. Significant interactions were further analyzed using post-hoc tests. Results: In pwPD, both COP pathway length (p < 0.01), ellipse area (p < 0.01) and mediolateral/anteroposterior root-mean-square (p < 0.01) were increased with the eyes closed, while the dual-task had no significant effect when compared to the single-task condition. Comparable results were observed in the control group for who COP pathway was longer in all conditions compared to eyes opened single-task (p < 0.01) and longer in conditions with eyes closed compared to eyes opened dual-task (p < 0.01). Similarly, all differences in EMG activity of pwPD were exclusively observed between eyes opened vs. eyes closed conditions, and especially for the forward leg’s soleus (p < 0.01) and backward tibialis anterior (p < 0.01). Conclusions: These results in pwPD without noticeable impairment of static balance encourage the assessment of both visual occlusion and dual-task conditions when the appearance of significant alteration during the dual-task could reveal the subtle worsening onset of the balance control. Parkinson’s disease (PD) is a neurodegenerative disease leading to plastic changes in the primary motor cortex, with a multifaced physiopathology, whose cardinal motor symptoms are bradykinesia, rigidity, rest tremor and postural instability . PosturaThe semi-tandem stance, a position where the toes of one foot are in line with the inside arch of the other foot, has been found to be the most challenging quiet stance task that can be sustained by healthy young and older subjects for at least 30 s  of PD coThis study was set to test how visual deprivation and dual-task affect body sway in OFF-state pwPD who hold a semi-tandem stance. A control group of healthy subjects underwent the same protocol. We expected an effect of both visual deprivation and dual-task on the semi-tandem performances, especially in the patients. We hypothesized a significant increase in neuromuscular activity of the leg muscles during the more challenging condition, in relation with an increase of ankle contribution to maintain body sways .Fifteen pwPD and fifteen healthy young adults  participAll subjects provided informed written consent as required by the Declaration of Helsinki. The experiment was approved by the local ethics committee of the University Paris-Saclay (EA4532) and by the “Comité de Protection des Personnes Ile-de-France XI” under identification number 19028-60429.Clinical assessments of pwPD were conducted at least 30 min before the study procedure. These tests included the Unified Parkinson’s Disease Rating Scale motor examination , Berg BaAll subjects  had to stand for 60 s on a force platform with their feet in a semi-tandem position  with theThe subjects completed three trials for each of the four randomly-assigned conditions: semi-tandem only with eyes open (EO-ST) and eyes closed (EC-ST), semi-tandem with additional cognitive task with eyes open (EO-DT) and eyes closed (EC-DT). In the eyes open conditions (EO-ST and EO-DT), they were instructed to fix the gaze on a 10 cm diameter target placed at eye level at 6 m distance.The subjects sat for 3 min between the trials to prevent them from extensive fatigue. The feet outline was drawn on the platform to ensure identical foot positions between all measurements. During the mentally idle condition, instructions such as “concentrate on the task” ,26, or “The ground reaction forces and moments were acquired using a floor-embedded force plate, sampling at 1000 Hz. Due to the inter-centeral study design the measurements were conducted with different systems but identical data collection settings: AMTI BP9001800  for the pwPD and a Kistler 9260AA  for the young adults.The positions of the center of pressure (COP) in the mediolateral (ML) and anteroposterior (AP) directions were computed offline using a custom-made MATLAB  routine and were bandpass filtered between 0.1 and 5 Hz  with a n2] . The PL was defined as the total length of the COP trajectory on the platform [mm]. MeanFreq came from the power spectrum of the COP sway in both AP and ML directions. The EA represents the area of the smallest ellipse which covered 95% of the COP samples [mm2] .Three non-linear methods—Sample Entropy (SampEn), Multi-Scale Entropy (MSE), and Multivariate Multi-Scale Entropy (MMSE)-were used to measure the regularity of the COP signals from the unfiltered signal and to compare their sensitivity between the incremented time series and the original time series. SampEN is essentially a negative logarithm of the conditional probability of the sequences of a data vector. If a vector of length N has repeated itself in tolerance γ for m points, it will also do so for m + 1 point. The conditional probability means the ratio of counts of repeated time of m + 1 point to that of m points. Thereby, high SampEN arises from a low probability of repeated sequences in the data. Higher SampEN means lower regularity and more complexity in the data. Based on SampEN, MSE is a method evaluating the complexity of signals over different time scales while MMSE generalizes the analysis to the multivariate case . In shorEMG activities were analysed for the M. tibialis anterior (TA), M. soleus (SOL), and M. Tensor of Fascia Lata (TFL). According to the respective leg positions, Muscle were determined as frontal and backward TA, SOL and TFL. Signals were recorded using a wireless EMG system in the pwPD  and in the young adults . The standard guidelines recommended by the SENIAM  were appp < 0.01 was selected to consider significant results.Descriptive statistics were computed for all variables under the four conditions. The normality of distribution (Shapiro-Wilk test) and homoscedasticity of data (Levene’s test) were verified before the application of the parametric tests. Since the criteria were not satisfied for all parameters, sway metrics were log-transformed to meet the normality request for further analysis. In pwPD and controls, the effect of vision privation, cognitive task, and vision X cognitive were judged by a 2 (eyes opened/eyes closed) × 2 (without/with cognitive task) repeated-measures ANOVA. Significant interactions were further analyzed using Bonferroni post-hoc tests. Also, a Bonferroni pairwise correction was applied to account for multiple comparisons  and a threshold of The characteristics of the pwPD and participants in the control group are displayed in p < 0.001, longer with eyes closed: EO-ST 1606 ± 1060 mm vs. EC-ST 2546 ± 930 mm, EO-DT 2105 ± 1749 mm vs. EC-DT 2874 ± 1776 mm), ellipse area , RMS of AP and ML amplitude . No differences were found between other conditions for the other parameters, including non-linear metrics. Regarding muscle activities, forward SOL and backward TA resulted in increased RMS with eyes closed than with eyes open (p < 0.01), while the RMS of backward SOL and forward TFL were higher during EC-DT than during EO-ST (p < 0.01). In addition, multiple regressions have been conducted with age, sex, UPDRS and Berg Balance Scale as potential confounders. No effects were observed.In pwPD , differep < 0.01, EO-ST 1654 ± 220 mm, EC-ST 1985 ± 223 mm, EO-DT 1793 ± 272 mm, EC-DT 2041 ± 332 mm) and longer with eyes closed compared to EO-DT (p < 0.0001). Ellipse area was larger with eyes closed compared to EO-ST (p ≤ 0.01). Differences were found in MSE for AP and ML amplitudes (p ≤ 0.01), demonstrating a lower regularity for the eyes closed conditions than during EO-ST. EMG activities did not differ across the four conditions.The analyses of the control group lead to differences in the COP pathway length and ellipse area. The COP pathway was longer in all conditions compared to EO-ST (No differences between pwPD and controls were found for the length of the COP pathway, MeanFreq AP and few conditions for MeanFreq ML and MedianFreq ML. The other parameters were significantly different between groups, with better outcomes for the controls .In this study we investigated challenging postural tasks in pwPD with no noticeable balance impairments, to determine whether, and to which extend, subtle balance disorders can be detected. The current protocol, where visual deprivation and dual-task paradigms were evaluated during semi-tandem stance, provided new insights. Overall trends in the results were similar in pwPD and young controls, although COP path length was significantly longer for controls when performing dual-task than single-task with the eyes opened. Maintaining postural stability involves sensorimotor transformations that continuously integrate several sensory inputs and coordinate motor outputs to muscles throughout the body . In thisThe lack of visual inputs implies a greater reliance on the integration of the other sensory outputs, with a suitable informational processing speed. A healthy person relies on visual, somatosensory, and vestibular information for balance control, but all or some of the components of this system may be dysfunctional in Parkinsonian patients . TherefoVuillerme and Nafati  observedOn the other hand, all difference in EMG activity have been exclusively observed between eyes opened vs. eyes closed conditions, especially for the forward leg’s M. soleus and backward M. tibialis anterior, which demonstrated increased RMS when eyes were closed. This result was expected based on the M. soleus’ fundamental support role during quiet standing , with a More surprising were the results under the dual-task condition. The pwPD included in the study demonstrated no alteration to biomechanical outcomes or EMG RMS when a cognitive task was added to the postural primary task, independently of the vision condition. The pwPD have issues in automating movements, which increases the attentional demand during daily activities and generates difficulties associating a simultaneous cognitive task to a motor task . NieuwhoIn the most complex task, under dual-task with eyes closed, the increase in both M. soleus and M. tibialis anterior activity of the backward leg might have reflected the necessity for the pwPD to use an ankle strategy with co-activation of the muscles and could underly the postural instability during quiet standing , since tIn addition to the most standard COP metrics, we analyzed non-linear parameters, such as entropy, to leverage information that traditional measurements do not reveal. While the use of non-linear methods has a long tradition to distinguish between visual conditions in elderly patients , former Some limitations must be discussed. The findings around dual-task are hard to interpret. First, the cognitive performance during the dual-task was not assessed and the trade-off between cognitive and balance tasks cannot be determined. Then, the findings may be due to the mild disease of the participants, or perhaps cortical control is not as important in early disease or compared to other motor tasks such as gait. One recent review suggests the importance of cerebellum for postural control . RegardiFuture studies should first overcome these limitations. The use of a conceptual model would be helpful to characterize patterns of cognitive-motor dual-task interference , especiaIn pwPD without noticeable impairment of static balance, visual deprivation seems to alter more standing performance during semi-tandem stance than the addition of a secondary cognitive task. However, for clinical assessment, it can be valuable to combine both visual occlusion and dual-task conditions, when studies in pwPD with higher functional impairments usually demonstrated significant poorer balance on dual-task than single-task . Appeara


After exclusion of values obtained from four patients with ECMO or Cytosorb® adsorber the median concentration was 0.91 µg/mL  in the RRT group. In addition to previous recommendations we propose to monitor ISA trough plasma concentrations in certain circumstances including RRT, other extracorporeal treatments and obesity.Isavuconazole (ISA) is a triazole antifungal agent recommended for treatment of invasive aspergillosis or mucormycosis. The objective of this study was to evaluate ISA levels in a real world setting in a mixed patient cohort including patients with non-malignant diseases and extracorporeal treatments, and to correlate findings with efficacy and safety outcomes. We investigated 33 ISA treatment courses in 32 adult patients with hematological and other underlying diseases and assessed the clinical response, side effects and ISA trough plasma concentrations. ISA treatment led to complete and partial response in 87% of patients and was well tolerated. The median ISA plasma concentration was 3.05 µg/mL  in patients without renal replacement therapy (RRT) or extracorporeal membrane oxygenation (ECMO) and significantly lower in patients with RRT including cases with additional ECMO or Cytosorb Isavuconazole (ISA) is a triazole antifungal agent recommended for treatment of invasive aspergillosis or mucormycosis ,2,3. In One larger study investigated 283 plasma concentrations from real world ISA use, and found ISA plasma concentrations of >1 µg/mL in >90% of patients, resembling findings from the SECURE trial . HoweverThe objective of this study was to evaluate ISA levels in a real world setting in a mixed patient cohort including patients with non-malignant diseases and extracorporeal treatments, and to correlate findings with efficacy and safety outcomes.This observational national multicenter cohort study prospectively included patients aged 18 years or older undergoing treatment with ISA at four centers in the Southern and Eastern part of Austria, the Medical University of Graz, the Landeskrankenhaus (LKH) Graz 2, LKH Hartberg, and the Klinikum Baden from November 2016 to October 2019.Based on recommendations of the local drug advisory committees ISA was allowed to be administered in patients with possible/probable/proven invasive aspergillosis using revised EORTC MSG criteria and/or Blot criteria in case of invasive aspergillosis in critical ill patients ,14. PatiPatients’ medical records were reviewed individually by using a standardized data collection template in order to collect demographic information and clinical data, mycological laboratory test results, ISA trough plasma concentrations, as well as ISA formulation, dosing information, termination of ISA treatment as well as other antifungal therapy, clinical response, serious adverse events, and breakthrough infections. Treatment success, failure and death were classified according to EORTC/MSG criteria . BreakthThe day of the first administration of ISA was considered as day 0. Samples for determination of ISA trough plasma concentrations were obtained in the morning immediately prior to scheduled ISA infusion or intake as ordered by the treating physician. As no routine TDM algorithm for ISA was available by the current literature ,6,7, plap-value of <0.05 was considered significant.The study was approved by the local ethics committee, Medical University Graz, Austria (protocol number 29–444 ex 16/17). All statistical analyses were performed using R version 3.5.1. Continuous data  are presented as medians (inter-quartile ranges [IQR]). The data at patient level were summarized by calculating the median  of Isavuconazole levels. More specifically, in patients with more than one single measurement, the median and maximum levels of Isavuconazole were calculated. By doing so, each patient is represented by one single summary measurement (median or maximum) and standard statistical tests can be applied. We used the median as the main variable for comparison between groups and the maximum value was used for sensitivity analysis. Measurements of patients assigned to specified groups  were considered independent. Group comparisons were performed by Wilcoxon-rank-sum test. A 2 . A total of 14 patients (44%) had hematological diseases , whereas the 18 other patients (56%) had mixed underlying diseases  male, nine (28%) female; median age 60 years, range 24 to 85 years, IQR 46–69). Study patients had a median body mass index of 24.6 kg/mdiseases .® adsorber therapy .Fourteen of 32 patients (44%) had proven invasive fungal disease, nine (28%) had probable and nine (28%) had possible invasive fungal disease. One patient with two courses of ISA was classified as suffering from probable invasive fungal disease on both occasions. The median duration of ISA administration was 45 days . In 12/33 (36%) ISA courses the patients received only the intravenous and in 6/33 (18%) courses only the oral formulation of ISA. In 15/33 (45%) ISA courses patients received the intravenous and oral formulation. The intravenous ISA formulation was used for a median of 13 days  whereas the tablet was used for a median of 70 days . In a total of 20/33 courses (61%) the patients received antifungal therapy or prophylaxis prior to ISA, in six of these 20 courses (30%) ISA was used due to failure and in 13/20 courses (65%) ISA was used due to side effects of preceding antifungals. A total of 11/32 patients (34%) were on renal replacement therapy , while three of these patients additionally had extracorporeal membrane oxygenation (ECMO) and one additionally had CytosorbFusarium solani blood stream isolate and died six weeks later . ISA plasma concentrations were 1.51 µg/mL on day 4, 1.32 µg/mL on day 6 of ISA treatment, and 1.36 µg/mL two days after termination of ISA treatment. In 3/33 (9%) ISA courses no classification was possible . Overall, a total of 11/32 patients (34%) had a fatal outcome during hospitalization including the three IFI related deaths. No breakthrough infection during ISA administration was observed.Complete response occurred in 18/30 (60%) ISA courses, partial response in 8/30 (27%), and stable disease in 1/30 (3%) ISA courses. Assessment was not possible in three cases. Three out of 32 patients (9%) had a fatal outcome during hospitalization attributable to fungal disease. One patient with pulmonal and cerebral aspergillosis died on day 4 and had no ISA plasma concentrations measured. One patient with possible invasive mould disease died on day 10 of ISA treatment and had 1.36 µg/mL ISA plasma concentration on day 3 (without RRT) and 1.69 µg/mL on day 6 while on continuous hemodialysis. The remaining patient received six days of ISA, was switched to lipAmpB plus voriconazole after receiving the antifungal susceptibility testing of a Six of 33 (18%) ISA treatments led to adverse events including one case with an anaphylaxis , one with leucopenia (1.52G/L), two with elevated liver enzymes, one with paraesthesia and one with erythema and elevated liver enzymes. All of these patients had concomitant medication, thus the role of ISA in the reported adverse events remains unclear. All patients recovered fully from adverse events.® adsorber therapy) the median concentration was 0.91 µg/mL  in the RRT group. Patients without extracorporal treatments also had higher maximum Isavuconazole trough levels compared to the groups with extracorporal treatments . Two patients (body mass index 23 and 26 kg/m2) with influenza and ARDS received ECMO  for pulmonary support and continuous RRT. One ECMO patient was treated with a standard dose of ISA for invasive aspergillosis since preceding and dose escalated intravenous voriconazole did not reach target levels of >1 µg/mL. On day 12 after initiation of intravenous ISA the plasma concentration was measured and was 1.79 µg/mL. The patient died 2 days later due to a gangrenous bowel. The other ECMO patient had a standard dose of ISA for treatment of probable intraabdominal Candida parapsilosis infection after failure of caspofungin. On day 1 and 4 after initiation of intravenous ISA the plasma concentration was 0.74 µg/mL (during loading dose) and 0.57 µg/mL, respectively. ECMO was terminated on day 6. Subsequent ISA plasma concentration during the second ISA treatment course of this particular patient was 2.44 µg/mL while receiving RRT. The patient died 5 weeks later due to secondary sclerosing cholangitis. The third patient was treated with a Cytosorb® cytokine adsorber within a continuous RRT circuit for 4 days and had an ISA plasma concentration of 1.3 µg/mL immediately prior and 0.82 µg/mL on the last day of adsorber treatment. Subsequent ISA plasma concentrations determined 14 and 32 days after the adsorber treatment but during ongoing RRT were 0.62 µg/mL and 0.91 µg/mL. This patient suffered from pulmonary invasive aspergillosis and had a favorable response after 56 days of ISA treatment. The fourth patient developed ARDS after major cardiac surgery and was treated for pulmonary invasive aspergillosis. ISA concentration was measured daily during a period of 18 days. During ECMO the median ISA concentration was 1.7 µg/mL. On day 12 additional RRT was initiated and ISA concentration decreased to 0.8 µg/mL. ECMO was discontinued on day 15 but RRT was still ongoing and ISA concentration remained below 0.9 µg/mL. Boxplots of ISA plasma concentrations with and without extracorporeal treatments are shown in A total of 145 ISA plasma concentrations were measured. Five ISA plasma concentrations were excluded due to erroneous sampling time points . Thus, 140 ISA plasma concentrations were analyzed from 29 courses of ISA treatment with a median of three measurements per patient (range 1–18). There was no change in the dosage due to the measured plasma levels. In three out of four patients with missing ISA plasma concentrations ISA was administered for less than 5 days  while it was administered for one month in one patient. Median time of first measurement after initiation was 2.28 days . The median plasma concentration was 2.35 µg/mL (range 0.66 to 9.1 IQR 1.49–3.71). After exclusion of values during RRT or ECMO the median ISA concentration was 3.05 µg/mL  . The medAmong the six of 33 (18%) ISA treated patients with adverse events, four had ISA plasma concentrations measured, which were always below 5.5 µg/mL .In our cohort ISA was used for treatment of invasive fungal diseases in patients with malignant and non-malignant underlying diseases. Overall, ISA was well tolerated compared to previous studies ,10. EighAs influenza might result in ARDS requiring extracorporeal membrane oxygenation (ECMO) and was reported to be an independent risk factor for invasive pulmonary aspergillosis  the selep < 0.001; 42% reduction rate of ISA concentration) [These findings are of course limited by low numbers but may highlight the need of ISA TDM in patients with ongoing ECMO, a fact that was also previously reported for other antifungals like voriconazole or caspofungin. Based on ISA concentrations measured in our ECMO patients one might assume that ISA is sequestrated within the ECMO circuit during the first days of treatment leading to low plasma concentrations according to an assumption previously described for voriconazole . As bothtration) . Our critration) .® cytokine adsorber four times for 16 h each within a RRT circuit. Whereas no data of this cytokine adsorber are available determination of ISA plasma concentrations in our patient suggest that this particular device might lower ISA plasma concentrations. However, as the patient subsequently showed ISA plasma concentrations of 0.68 µg/mL and 0.91 µg/mL 14 and 32 days after cytokine adsorption the exact effect of the Cytosorb® cytokine adsorber remains unclear.Cytokine reduction by hemoadsorption aims to attenuate the overwhelming systemic levels of pro-inflammatory and anti-inflammatory mediators released in the early phase of sepsis. One of our ISA treated patient had a Cytosorb2 (140 kg body weight) which was outside the BMI and weight standard deviations of published ISA data [2 the patient received the standard dose of ISA for treatment of probable invasive aspergillosis and was closely monitored by ISA plasma concentrations. The obtained ISA plasma concentrations were 1, 2.42, 3.68 and 3.42 µg/mL, respectively. The patient had a complete response.One of our patients had a body mass index (BMI) of 39.6 kg/mISA data ,24. PrevISA data . DespiteA post hoc analysis of ISA exposure-response relationship for measures of efficacy and safety in patients with invasive aspergillosis and infections by other filamentous fungi from the SECURE clinical trial showed that no statistically significant relationship between ISA exposure and either efficacy or safety endpoints. The authors concluded that lack of association between exposure and efficacy indicates that the ISA exposures achieved by clinical dosing were appropriate for treating the infecting organisms in the SECURE study and that side effects were not related to increase in exposures . ComparaIn summary, ISA was used in patients with and without hematological malignancies for treatment of invasive fungal diseases. ISA was efficacious, well tolerated and showed ISA plasma concentrations comparable to previously successfully treated patients even in cases with high body weight (BMI). Plasma concentrations in patients receiving RRT were significantly lower compared to patients without. However, due to low numbers of patients with high body weight and RRT as well as inconsistent or low levels obtained in patients with ECMO or cytokine adsorber we propose to monitor ISA plasma concentrations in such patients to attain previously suggested but still uncertain target levels of ISA >1 µg/mL ,9,22.


Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from  Ion ce signal ,2, whilee signal . At the e signal ,7, malfue signal ,9, and de signal .The artificial lipid bilayer recording technique measures the ion transport activities of channel proteins along with other ion transporting proteins and peptides . This teThe physical processes that occur during lipid bilayer formation have been extensively studied and are described in standard textbooks . PrincipMembrane proteins, including channel proteins, are generally not inserted into bilayers directly from the aqueous solution, with certain exceptions such as voltage-dependent anion channels (VDACs). The most efficient method used to incorporate channel proteins into bilayers is vesicle fusion. However, because vesicles stochastically fuse with the bilayer, the process cannot be controlled, and thus sometimes takes a long time.Streptomyces lividans (KcsA) or from Methanobacterium thermoautotrophicum (MthK) channels by binding them to cobalt affinity gel beads with a histidine tag and then forming a lipid bilayer membrane with the channels on the bead by pushing the lipid layer onto the bead [In order to improve the measurement efficiency, several approaches have been proposed. For example, bilayer membranes can be promptly made in a lipid solution at the droplet–droplet interface ,14,15, dthe bead . In anotthe bead . By contthe bead ,20,21. In general, we have developed techniques for channel current recordings using various types of substrate-supported bilayers to improve the measurement efficiency of conventional artificial bilayer methods. Here, we report further enhanced efficiency by simplifying the bilayer formation technique and shortening the required time for the measurement preparation. This component technology could potentially lead to the miniaturization of a measurement apparatus. 2+ affinity gel beads  from Clontech , and Sepharose 4B beads were from Cytiva Japan . All other chemicals were commercial products of analytical grade.Asolectin, alamethicin, and gramicidin were purchased from Sigma , α-hemolysin was from Abcam plc , CoStreptomyces lividans (KcsA channel) gene cloned into pQE-30 vectors, including an N-terminal hexahistidine tag, was a gift from Dr. Kubo of the National Institute of Advanced Industrial Science and Technology. We used a KcsA mutant (E71A) made by using the QuickChangeTM site-directed mutagenesis kit (Stratagene), because the E71A mutation is known to inhibit inactivation.The cDNA of mouse voltage-dependent anion channel 1 (VDAC1) (ENSMUST00000102758.7) was subcloned into a multi-cloning site (NdeI-XhoI) of the pET21b vector. The potassium channels from Escherichia coli BL21(DE3) and then overexpressed in the presence of 1 mM isopropyl-d-thiogalactopyranoside (IPTG) for 4 h. The BL21(DE3) pellets were incubated in BugBuster Protein Extraction Reagent  containing protease inhibitors  and benzonase (Merck) for 5 min at room temperature, and then incubated in BugBuster Protein Extraction Reagent containing 250 μg/mL lysozyme (Merck) for 5 min at room temperature. After they were centrifuged at 16,000× g for 20 min, the precipitates were sonicated in binding buffer ) and then centrifuged at 16,000× g for 10 min. The supernatant was mixed with nickel resin  and rotated at 4 °C for 60 min. Nonspecific bound proteins were removed with 5-fold volume of binding buffer, 5-fold volume of wash buffer ), and 5-fold volume of refolding buffer , 50 mM Tris-HCl (pH 8.0)). Then, VDAC1 was eluted with elution buffer ) and dialyzed with a dialysis membrane . VDAC1 protein was further purified by cation exchange using ÄKTA explorer 10S  and RESOURCE™ S column .The pET21b plasmids containing the VDAC1 sequence were transformed into Escherichia coli XL1-Blue and overexpressed by the addition of IPTG to a final concentration of 0.5 mM for 2 h. The expressed channels were extracted from membrane fractions by 10 mM n-dodecyl-d-maltoside . Co2+ affinity gel beads  equilibrated with normal buffer , 100 mM KCl) were added to the extracted channel protein solution and incubated for 30 min at 4 °C in order to bind histidine tags to the gel beads. Nonspecific bound proteins were removed with washing buffer . The pQE-30 plasmids containing the KcsA sequence were transformed into g. The supernatant was centrifuged for 40 min at 14,000× g and then for 20 min at 9500× g. The pellet was resuspended in solution  with a Teflon pestle homogenizer. Samples were incubated on ice for 1 h and centrifuged for 1 h at 42,000× g. The pellets were resuspended in 400 mM KCl, 10% sucrose (w/w), and 5 mM Na-PIPES, pH 6.8. The microsomes were placed on top of a discontinuous sucrose gradient (w/w): 20:25:30:35:40%. The gradient was centrifuged at 67,500× g in a swinging rotor for 18 h. Membranes obtained from the 20:25% sucrose interface were used in this study.Myometrium plasma membrane vesicles containing BK channels  were prepared according to the method of Toro et al. . Membran2+ affinity gel bead, on which KcsA proteins were immobilized, was used in the case of KcsA channel recordings and a Sepharose 4B bead was used for other types of channels. A glass capillary  was pulled to make a pipette with a very fine tip (<1 μm) using a pipette puller , then the tip was cut to make a large aperture (>10 μm). The tip was fabricated using a microforge  in order to smooth the surface. 2+ affinity gel bead fixed at the tip of a glass pipette by suction. The glass pipette was set with a pipette holder and a bead was fixed at the tip of the pipette by suctioning it through a tube from the holder with a syringe. In A hydrogel bead was immobilized to the aperture at the glass pipette tip by suction. A Con-decane or hexadecane) was layered over the recording solution layer. Bilayers were made by moving the bead fixed at the pipette tip through this lipid solution layer into the recording solution  by an Ag–AgCl electrode defined the membrane potential. Unless otherwise noted, the signal was filtered at 1 kHz, sampled at 10 kHz, digitized, then stored on a PC. One of two channel forming peptides , the hemolytic protein α-hemolysin, or detergent-solubilized VDAC1 was added to the recording solution at appropriate concentrations before the bilayer formation. These molecules were spontaneously incorporated into the bilayer formed on the Sepharose 4B bead to make ionic channels. KcsA channel proteins were spontaneously incorporated into the bilayers by forming bilayers with a Con-decane). The aqueous recording solution was extruded out from a polyethylene tube (0.1–0.5 mm inner diameter) placed near the bead by manually exerting positive pressure with a syringe, such that it contacted the bead in the lipid solution. The contact was observed with a microscope. A bilayer membrane was promptly formed at the water–gel interface. The solution in the tube was held at virtual ground level so that the voltage at the solution level in the glass pipette connected to a patch clamp amplifier by an Ag–AgCl electrode defined the membrane potential , which agreed well with the value obtained by conventional methods [ methods .n = 5). The current fluctuation showed voltage dependency, as reported in previous papers [s papers . The opeStaphylococcus aureus, which is known to form a heptamer in bilayers, forming a large ion channel pore [n = 5), which was identical to the values obtained by conventional bilayer methods [nel pore ,28. The  methods , again vn = 5), and the channel showed voltage dependent gating as reported previously [eviously . The above confirmed that our method can be used to measure the currents of several types of channels. Importantly, the channel current properties observed were in good agreement with the results obtained from other techniques, such as the conventional planar bilayer technique and patch clamp analysis. Thus, our method can be used to study a wide range of channels. Moreover, the channel current measurements were relatively easy, as we only needed to form bilayers with recording solutions containing the channel peptides or proteins. All of the channels shown in Streptomyces lividans. It is one of the best-characterized channel proteins because its structure is very simple. As described in the Materials and Methods section, purified KcsA channels were solubilized with detergent and immobilized on a gel bead via histidine tags. The bead was moved to contact the aqueous solution through the lipid solution layer, and immobilized KcsA channels were reconstituted immediately after the bilayer formation.Next, we immobilized physiological channel proteins (which cannot be directly inserted into bilayers from an aqueous solution) on the surface of a hydrogel bead and reconstituted them into bilayers to measure their currents. As we previously reported, some types of physiological channel proteins that have been solubilized with detergent and immobilized on a gel  or solid+ channel purified from pig uterus. Prior to the bilayer formation, a hydrogel bead immobilized at the glass pipette tip was dipped into a membrane vesicle suspension that contained BK channels in the vesicular membrane. Immediately after contact between the gel and lipid–water interface, the current began to fluctuate, showing that BK channels were inserted into the bilayer by vesicle fusion. It is likely that the incorporation rate was high compared with conventional bilayer methods, because the space between the gel and the lipid was narrow, meaning that the vesicle concentration was kept high. In Physiological channel proteins were also measured by incorporating them into bilayers through vesicle fusion. Bilayers were promptly made by pushing out the aqueous solution from a fine tube, such that the solution contacted a gel bead. The effect of this strategy is to miniaturize the apparatus. Above, we showed that we could measure the properties of various types of channels by using bilayers formed on a gel bead. Although our technique makes the recordings more efficient than the conventional bilayer technique, we should develop a simultaneous parallel recording technique to further increase the measurement efficiency. However, because our apparatus contains a bulky manipulator, it is not suitable for the parallel recordings of many channels. Therefore, we modified the bilayer formation technique to miniaturize the apparatus.Next, we used this modified bilayer formation technology to perform simultaneous recordings of different types of channels. The current recordings shown in By improving our previously developed channel recording techniques, here we developed an efficient method for measuring ion channel activities. We measured the natural properties of various types of channels incorporated into gel-supported bilayers. We were able to simultaneously measure different types of channels in different environments. This method allows miniaturization of a recording system. In the future, by making this system multi-channel, we may obtain a high-throughput device for channel current measurement.


Systemic CD8+ T cell depletion abolished NLGP-mediated metastasis inhibition and reappeared upon adoptive transfer of NLGP-activated CD8+ T cells. Interferon γ-secreting from CD8+ T cells inhibit the expression of angiogenesis regulatory vascular endothelial growth factor and transforming growth factor β and have an impact on the prevention of colonization. Neem leaf glycoprotein modulates dendritic cells (DCs) for proper antigen presentation by its DC surface binding and upregulation of MHC-I/II, CD86, and CCR7. Neem leaf glycoprotein–treated DCs specifically imprint CXCR3 and CCR4 homing receptors on activated CD8+ T cells, which helps to infiltrate into metastatic sites to restrain colonization. Such NLGP's effect on DCs is translation dependent and transcription independent. Studies using ovalbumin, OVA257−264, and crude B16F10 antigen indicate MHC-I upregulation depends on the quantity of proteasome degradable peptide and only stimulates CD8+ T cells in the presence of antigen. Overall data suggest NLGP inhibits metastasis, in conjunction with tumor growth restriction, and thus might appear as a promising next-generation cancer immunotherapeutic.Neem leaf glycoprotein (NLGP), a natural immunomodulator, attenuates murine carcinoma and melanoma metastasis, independent of primary tumor growth and alterations in basic cellular properties . Colonization event of invasion–metastasis cascade was primarily inhibited by NLGP, with no effect on metastasis-related invasion, migration, and extravasation. High infiltration of interferon γ (IFN-γ)–secreting cytotoxic CD8 Metastasis, an eminent hallmark of cancer , is an i+ T cells via influencing maturation of dendritic cells (DCs)] primarily intervenes two hallmarks of cancer, that is, “avoiding immune destruction” and “angiogenesis” (Neem leaf glycoprotein (NLGP) therapeutically restricts melanoma, carcinoma, and sarcoma solid tumor growth –15 by imgenesis” , 7 of di+ T cell depleting antibody (clone 2.43) was purchased from Taconic .RPMI-1640, Dulbecco modified Eagle medium (DMEM)-high-glucose, heat-inactivated fetal bovine serum (FBS), tri-reagent, and carboxy-fluorescein-diacetate succinimidyl ester (CFSE) were purchased from Invitrogen . CD4–fluorescein isothiocyanate (FITC)/Cy-chrome, Foxp3-phycoerythrin (PE) monoclonal antibodies (mAbs), and Cytofix/Cytoperm kit were procured from BD Pharmingen . Anti-CD25 (PE), anti–MHC-I , anti-MHC-II (PE), anti-CD80 (PE), anti-CD86 (PE), anti-CCR7 (PE), anti-CD44 (PE), and anti-CD69 (FITC) were purchased from Biolegends . Purified anti–mouse transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and interferon γ (IFN-γ) mAbs, secondary antibodies either fluorescence or peroxidase labeled, were obtained from e-Biosciences . Cytotoxicity detection kit [based on lactate dehydrogenase (LDH) release] Cyber-Green were procured from Roche Diagnostics . Aminoethylcarbazol (AEC) chromogen solution and aqueous mounting media were purchased from VECTOR Laboratories Inc. . Reverse transcription–polymerase chain reaction (RT-PCR) primers and cDNA synthesis kit were procured from MWG-Biotech AG  and from Fermentaus . DAPI-shield, peroxidase secondary antibodies were purchased from Sigma . Different inhibitors were procured from Calbiochem-Merck-Millipore and Sigma. CD8Azadirachta indica) leaves of same size and deep green color (indicative of same age), plucked from neem trees , were shed-dried and pulverized. Permission was taken to use such natural product from the National Biodiversity Authority (NBA), an autonomous and statutory body of the Ministry of Environment and Forests, Government of India (ref no. NBA/Tech Appl/9/518/12/14-15/4587). Leaf powder was soaked overnight in phosphate-buffered saline (PBS), pH 7.4; supernatant was collected by centrifugation at 1,500 revolutions/min (rpm). Crude neem leaf preparation was then extensively (four to five times) dialyzed against PBS, pH 7.4, and concentrated by Centricon membrane filter  with 10-kD molecular-weight cutoff. The purity of NLGP was checked by size exclusion–high-performance liquid chromatography (SE-HPLC) in a protein PAK 300 SW column of 7.5 mm (ID) ×30 cm. The protein peaks were determined by absorption at 280 nm in a UV recorder. Batch consistency of the NLGP is checked by HPLC and tumor growth restriction assay as per standard protocol.Mature neem  were obtained from the animal facility of CNCI, Kolkata. Autoclaved dry pellet diet  and water were given l-glutamine and penicillin (50 U/mL), and streptomycin (50 μg/mL) at 37°C with 5% CO2 and 95% humidity. As the cell lines were obtained from cell bank, cell authentication was not done by us.B16F10 (melanoma) cell line was obtained from the National Center for Cell Sciences, Pune, India, and LLC (carcinoma) cell line was obtained from ATCC, Manassas VA, USA. Cells were cultured in complete DMEM high-glucose media supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM 5) cells subcutaneously on the right flank of mice. After palpable tumor formation, one group of mice was subcutaneously injected with NLGP (1 μg/g of body weight/injection); the other group was treated with PBS weekly for 4 weeks in total or once only as per the experimental requirement. Solid tumor growth (in mm2) was measured using the formula: (width × length). Tumor-bearing mice were euthanized by overdose of ketamine HCL (160 mg/kg) + xylazine (20 mg/kg) intraperitoneally (i.p.) as per Committee for the Purpose of Control and Supervision of Experiments on Animals guideline when tumor size reached 20 mm in either direction or the animal looks sick or any necrosis of tumor was occurred. The animal's health was monitored every day.Solid tumors were developed in C57BL/6J mice by inoculation of B16F10 or LLC (5 × 105 cells) were injected through tail-vein (t.v.). Following tumor inoculation, mice were subcutaneously injected with NLGP (1 μg/g of body weight/injection) weekly for 4 weeks in total, keeping a PBS-treated group as control.For the development of lung metastasis, B16F10 or LLC cells . Fixed cells were stained with primary fluorescence antibody, mounted with fluoroshield-DAPI , and observed under fluorescence-microscope [LeicaDM4000B  or Olympus BX53 ] or confocal microscope .Cells were harvested by cytospin  on poly-Western blot of IFN-γ, granzyme B, TGF-β, VEGF, and cleaved caspase 3 was performed as described . Lungs wFlow-cytometric analysis was done as described . The datCells were stained according to the protocol given by the manufacturer, BD Pharmingen. Cells were washed twice with cold PBS and then resuspended in 1× binding buffer. Annexin V–FITC and propidium iodide (PI) (5 μL of each) were added and mixed gently with vortexing. Cells were incubated for 15 min at room temperature (25°C) in the dark followed by resuspending the cells with 400 μL of 1× binding buffer. Cells were analyzed immediately by flow cytometry.Cells were harvested, fixed in chilled 70% ethanol with vortexing, followed by incubation at −20°C overnight. Cells were washed twice with PBS containing 0.1% sodium azide, followed by 50 μL (100 ng/mL) RNase treatment. Cells were stained with 200 μL (50 μg/mL) PI to analyze flow-cytometrically immediately.Ki67 staining was done as described . In brieDifferent protumorous cytokines from tumor conditioning media  and cell supernatant from DC:T cell coculture  were measured  by enzyme-linked immunosorbent assay (ELISA) using microplate reader .Cellular total RNA was isolated using Trizol . Random hexamers were used to generate corresponding cDNA according to the manufacturer's protocol . Amplification was performed using 2× green mix . Amplification of the target genes was done by gene-specific primers as described . Primer A scratch was made with a scratcher on confluent B16F10 cells, followed by NLGP treatment (1.5 μg/mL). Wells were photographed at different time points to check the healing of wound (scratch).4 and 2 × 104 cells for migration and invasion, respectively) in serum-free media in presence or absence of NLGP. Migration or invasion was measured against the 10% FBS containing media for 12 h. Following incubation, cells were fixed with 2% paraformaldehyde and stained with 0.01% crystal violet. Cells in the upper chamber were removed by wiping with cotton swabs. Serum-free gradient was used as a negative control.Overnight serum-starved B16F10 or LLC cells were seeded in the upper chamber of either Transwell or BD invasion chamber (4 × 105) cells were adoptively transferred through t.v. injection. Lungs were harvested at desired time points and digested with collagenase (1.5 mg/mL) and DNase I (0.1 mg/mL) for 30 min at 37°C for single-cell preparation, and CFSE+ cells were analyzed by flow cytometry. In a separate set, harvested lungs were prepared for cryosectioning by standard method as described (B16F10 or LLC cells were stained with CFSE (5 mM) according to the manufacturer's protocol. Tumor  were either cocultured with DCs or transferred adoptively in mice.CD8tic lung  with the+ T cell depletion status in peripheral blood was monitored by flow cytometry.Tumor-bearing mice were peritoneally injected with CD8-depleting antibody (100 μg/50 μL) 24 h prior to NLGP administration on each time point. CD8+ T cells were isolated by magnetic bead–based positive selection (+ T (2 × 105) cells were adoptively transferred through t.v. injection.Metastatic lungs were harvested from PBS- and NLGP-treated mice at desired time points  and digeelection . Isolate+ T cells were isolated from PBS- and NLGP-treated lungs. Cellular cytotoxicity of those CD8+ T cells was checked by measuring LDH release assay according to the manufacturer's protocol (Roche Diagnostics). For antigen restimulation assay, CD8+ T cells were restimulated, and secreted IFN-γ was measured by ELISA. Assay was performed by the method as described (CD8escribed .Evans blue solution (0.1% in PBS) was injected through t.v. After 30 min of incubation, mice were sacrificed, and macroscopic observation was made.6 cells/mL) were cultured with complete RPMI-1640 medium containing 10% (vol/vol) heat-inactivated FBS, 2 mM l-glutamine, and Pen-Strep , with recombinant mouse Granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (10 ng/mL) and recombinant mouse Interleukin 4 (rmIL-4) (5 ng/mL) at 37°C in 5% CO2 for 6 days. Media including the supplements was refreshed every third day. On day 6 of culture, non-adherent cells were harvested and considered as immature bone marrow–derived DCs (BmDCs).A single-cell suspension was obtained after flushing bone marrow from tibia and femurs. Erythrocyte lysed (by ACK lysis buffer) cells  after filtration with 0.22-μm membrane filter .B16F10 or LLC cells were cultured in complete DMEM at 37°C in 5% COAcid stripping of MHC-I was done according to the method described . Then MHImmature DCs were stained with MHC-I and then incubated at 37°C in presence or absence of NLGP, keeping controls at 4°C. After 12 h, surface MHC-I was removed by acid stripping, and internalized MHC-I was measured flow-cytometrically.Different inhibitors MG132 (0.5 μm), chloroquine (50 μm), ammonium chloride (10 mM), brefeldin A (10 nM), cytochalasin D (10 μm), cytochalasin B (20 μm), bortezomib (1 ng/mL), leupeptine (100 μm), dimethyl amilorite (200 μm), and cycloheximide (5 μm) were used to pretreat BmDCs. B16F10, LLC lysates, ovalbumin, and SIINFEKL were also used for different assays.7 cells/mL) in serum-free DMEM (measured by trypan blue dye exclusion assay) were lysed by 6 or 3 freeze (in liquid nitrogen)–thaw (at room temperature) cycles for lysate or necrotic cell preparation, respectively. The lysate was centrifuged at 15,000 rpm for 30 min at 4°C, and supernatant was collected. Total protein concentration was measured using Folin's phenol reagent. Necrotic cells were centrifuged at 2,500 rpm for 5 min, and collected cells were used in different experiments.Viable B16F10 or LLC cells (1 × 105 cells/mL), after treatment with NLGP and cell lysate (for 24 h), were fixed with 0.008% glutaraldehyde, washed with PBS, and then cocultured with autologous purified CD8+ T cells (1 × 106 cells/mL) in a total volume of 200 μL in complete RPMI-1640 media for 7 days in a 96-well plate. Cells and supernatants were collected for further analysis.Dendritic cells  or three to six (for in vitro assays) independent experiments. Statistical significance was established by unpaired Student t-test (for two groups) or one-way analysis of variance followed by Tukey post-hoc test (for more than two groups) using GraphPad Prism 5 software . For survival study, Kaplan-Meier survival analysis followed by data analysis with log-rank (Mantel-Cox) test was used. Differences between groups attaining a p-value of 0.05 are considered as significant.ImageJ  was used for image analysis, brightness–contrast adjustment, quantification of agarose gels, manual cell counting for invasion/migration assay, and measurement of wound healing. Quantity one (Bio-Rad) was used for Western blot gel scanning and Image Studiolite  was used for Western blot analysis. All reported results represent the mean ± SEM of data obtained in either one  in C57BL/6J mice keeping PBS-treated mice as control . In addiConsidering the linear relationship between primary tumor volume and metastasis, we checked the impact of NLGP-mediated primary tumor reduction on metastasis development. Accordingly, we used the experimental metastasis model (EMM) where tumor cells were inoculated intravenously without developing any primary tumor. On treatment completion , we founin vitro NLGP treatment has no direct effect on tumor cells or their secretomes  were injected t.v.  tumor cells were inoculated t.v., and significantly fewer number of tumor cells were detected in the lungs compared to the PBS-treated group  assay revealed higher IFN-γ release from the same T cells of the NLGP-treated mice group only  into a separate mice cohort, already inoculated with B16F10 cells  cells were collected after 48 h of NLGP treatment and found higher per-cell MHC-I expression along with increased CD80 and CCR7 in comparison to control  on purified CD8+ T cells after DC:T (1:10) cell coculture  enhanced NLGP-induced MHC-I expression. As phagosome and lysosome fusion gets inhibited by chloroquine and NH4Cl, it creates opportunity for antigen within phagosome to get export into cytosol for proteasome-mediated degradation. Hence, this leads to increase in total pool of proteasome-derived peptide, which in turn augments in the NLGP-mediated MHC-I increment. Cysteine protease inhibitor, leupeptin, which inhibits the cathepsin family protein within the phagolysosome, probably does not aid in antigenic protein export within cytosol, and hence does not augment NLGP-mediated MHC-I upregulation to a significant level. Phagocytosis inhibitors, cytochalasin D and cytochalasin B, and macropinocytosis inhibitor, dimethyl amiloride, did not show any effect (4Cl) augments this upregulation and indicates the possible involvement of NLGP in cross-presentation as cross-presentation is very relevant in tumor biology, where extracellular proteins are presented in the context of MHC-I.As NLGP upregulates MHC-I, next we wanted to know how alterations in antigen processing and presentation affect this upregulation. We found that in the presence of proteasome inhibitors, MG132 and bortezomib, NLGP-mediated MHC-I upregulation was inhibited. This observation clearly suggested that, in the absence of proteosome-generated peptide, NLGP treatment does not increase the surface expression of MHC-I and hence does not alter the basic principle, which states that only antigen-peptide–bound MHC-I gets exported to the cell surface. Protein transport inhibitor, brefeldin A, also inhibits NLGP-mediated MHC-I upregulation, which is also in line with the basic principle of cellular export, whereas phagosome and lysosome fusion inhibitor  as a foreign antigen. Two different antibodies were used; one specifically detects SIINFEKL-bound H2Kb, and the other detects antigen-bound H2Kb/H2Db. More MHC-I expression was detected by H2Kb/H2Db-specific antibody in the NLGP-treated group, whereas slight increment was detected by SIINFEKL-specific H2Kb . Apart from CD8+ T cell–dependent killing, we macroscopically detected reduced angiogenesis in the tumor and its adjacent areas; those are which were in line with the previous observation (+ and VEGFR2+ cells), along with the decreased level of angiogenesis regulatory factors such as VEGF and TGF-β, was detected in metastatic lungs after NLGP treatment. This may have an indirect role in metastasis attenuation. However, this study does not demonstrate the particular mechanism of reduced angiogenesis, but immune-mediated (mainly by IFN-γ) angiogenesis inhibition is already documented by us , and further elucidation of the signaling pathway is ongoing. T cell activation by dectin-1–stimulated DCs has been reported by others in context to fungal infection; however, the literature on antitumor role of dectin-1–activated DCs is very limited –48. Leibry tumor , 45. The antigen . Overallic drugs , 50. On + T cell dependence of NLGP action. Preclinical exploration of human tumor model specifically demands the use of nude or SCID mice, and those are immune compromised and lack CD8+ T cells, in particular.Validation of NLGP's effect in preclinical human tumor models is not possible because of CD8+ T cells, which skews the ongoing dialogue between the immune system and cancer cells to prevent the primary tumor growth and metastatic colonization. Based on our accumulated data from present or previous studies, we are in good faith that adjuvant and/or neoadjuvant treatment with NLGP in combination with other immunotherapy and/or chemotherapy will exhibit synergistic benefits in clinical outcome.A conglomeration of all these results proves that NLGP, via altering maturation of DCs, generates antigen-specific activated CD8Additional Files). Raw data related to this publication are available from the corresponding author on reasonable request.The data and material related to the findings of this study are presented in main figures within the article or as supplementary information files .ABh, ABo, and RB designed the study, analyzed the data, and wrote the manuscript. ABh, IG, and NG performed the research. AS and SD performed PCR and WB. AD performed confocal microscopy. PN, SG, TG, EH, and SB provide resources. ABo and RB supervise the project. RB acquired the fund. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Loss of smell and taste are commonly reported symptoms associated with coronavirus disease 2019 (COVID-19); however, the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in people with acute loss of smell and/or taste is unknown. The study aimed to determine the seroprevalence of SARS-CoV-2 antibodies in a community-based population with acute loss of smell and/or taste and to compare the frequency of COVID-19 associated symptoms in participants with and without SARS-CoV-2 antibodies. It also evaluated whether smell or taste loss are indicative of COVID-19 infection.n = 392) of participants were female. A total of 567 (96.1%) had a telemedicine consultation during which their COVID-19–related symptoms were verified and a lateral flow immunoassay test that detected SARS-CoV-2 immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies was undertaken under medical supervision. A total of 77.6% of 567 participants with acute smell and/or taste loss had SARS-CoV-2 antibodies; of these, 39.8% (n = 175) had neither cough nor fever. New loss of smell was more prevalent in participants with SARS-CoV-2 antibodies, compared with those without antibodies , whereas taste loss was equally prevalent . Seropositivity for SARS-CoV-2 was 3 times more likely in participants with smell loss  compared with those with taste loss. The limitations of this study are the lack of a general population control group, the self-reported nature of the smell and taste changes, and the fact our methodology does not take into account the possibility that a population subset may not seroconvert to develop SARS-CoV-2 antibodies post-COVID-19.Text messages, sent via primary care centers in London, United Kingdom, invited people with loss of smell and/or taste in the preceding month, to participate. Recruitment took place between 23 April 2020 and 14 May 2020. A total of 590 participants enrolled via a web-based platform and responded to questions about loss of smell and taste and other COVID-19–related symptoms. Mean age was 39.4 years (SD ± 12.0) and 69.1% , an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, was declared a pandemic in March 2020.COVID-19 can cause loss or reduced ability to smell (anosmia) or taste, without cough or fever, but few countries recommend self-isolation and testing on the basis of smell or taste changes alone.This study aimed to find out the proportion of people who have developed SARS-CoV-2 antibodies in a community-based population with a newly developed loss in their sense of smell and/or taste in London, UK.Text messages were sent out to people registered with a number of primary care centers in London inviting people with a new loss in their sense of smell and/or taste to participate.Recruited participants completed online questionnaires regarding demographics, their loss of smell and/or taste, and other COVID-19 symptoms, before they had a telemedicine consultation with a healthcare professional who confirmed the history of their symptoms and supervised a test to find out if they had SARS-CoV-2 antibodies.A total of 78% of 567 people with smell and/or taste loss had SARS-CoV-2 antibodies; of these, 40% had neither cough nor fever, and participants with loss of smell were 3 times more to have SARS-CoV-2 antibodies, compared with those with loss of taste.Loss of smell is a highly specific symptom of COVID-19.COVID-19 can present with loss of smell and/or taste without cough or fever.Loss of smell should be take into consideration in case isolation, testing, and treatment strategies for COVID-19. Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has spread exponentially, with 27,417,497 cases and 894,241 deaths reported from 216 countries by 9 September 2020 ”. Participants were then directed to an online platform  with the study information and an eligibility check. Inclusion criteria were age ≥18 years, proficiency in written and spoken English, and access to video-calling. Exclusion criteria were any preexisting loss of the sense of smell or taste of longer than a month’s duration. Participation was voluntary, and written informed consent was obtained electronically. Enrolled participants completed an online questionnaire see , capturiParticipants were subsequently sent a point-of-care testing kit detecting the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to SARS-CoV-2. A healthcare professional arranged a telemedicine video consultation with each participant. At the beginning of the consultation, they asked the participant to describe the changes that they had experienced in their sense of smell and/or taste. Participants were then supervised in performing the antibody test using a finger-prick sample of whole blood, and their results were discussed with them. Photographs of the test cassette were obtained and reviewed independently by a second healthcare professional to confirm the result. Participants with a prior COVID-19 PCR test result were offered antibody testing irrespective of their result. Testing was carried out between 24 April 2020 and 22 May 2020.The antibody test used detects the presence of IgM and IgG antibodies to SARS-CoV-2 via lateral flow immunoassay  . As partThe study received ethical approval from the National Health Service Queen’s Square Research Ethics Committee  and was conducted in line with the declaration of Helsinki and Good Clinical Practice.https://www.graphpad.com/scientific-software/prism/) and STATA version 15 (https://www.stata-uk.com/). Data analysis was planned on completion of SARS-CoV-2 antibody testing. There was no prospective study protocol or analysis plan, and no data-driven changes to analyses took place. Descriptive analyses included the calculation of means (plus standard deviation [SD]) for continuous variables and numbers  for categorical variables. Chi-squared tests were performed on categorical data as part of the secondary analysis. The significance level was adjusted for multiple comparisons by applying a Bonferroni correction when comparing symptoms other than loss of smell or taste. Logistic regression analysis was performed to estimate the association between loss of smell and/or taste and the presence of SARS-CoV-2 antibodies.A sample size calculation was undertaken in order to determine the study’s recruitment target, using the information on reported symptoms from the web-based COVID symptom study app developed by King’s College London and symptom reporting between the 24–29 March 2020 . To calcn = 392) were female, 30.5% (n = 173) male, and 0.4% (n = 2) of other sex. A total of (n = 311) 79.3% of female participants had a positive test result, compared with 73.4% (n = 127) of male participants (p = 0.120).A total of 33,650 text messages were sent out to people registered with 4 participating primary care centers. A total of 650 participants completed the registration process; 60 participants were ineligible and excluded. The participant flowchart  is illusn = 531) reported loss of their sense of smell. This was described as complete smell loss by 69.9% (n = 371) and as a partial smell loss by 30.1% (n = 160). No smell loss was reported in 10.0% (n = 59). A total of 89.8% (n = 530) of participants reported loss of their sense of taste. This was described as complete by 47.4% (n = 251) and as partial by 52.6% (n = 279). No taste loss was reported in 10.1% (n = 60) of participants. Combined loss of smell and taste was reported by 80.0% (n = 472).Among the 590 participants who completed questionnaires, 90.0% (n = 439) had a positive SARS-CoV-2 result . One further participant was included with a positive PCR result (77.6% [n = 440] positive). Participants with and without SARS-CoV-2 antibodies were comparable in terms of age, sex, ethnicity, smoking status, and frequency of other reported symptoms (n = 229) of the participants with SARS-CoV-2 antibodies had no history of cough, and 39.8% (n = 175) had neither a fever nor a cough.Of 590 eligible participants, 567 participants (96.1%) underwent SARS-CoV-2 testing. A total of 77.4% (symptoms . Importan = 567), 89.9% (n = 510) reported loss of their sense of smell and 89.7% (n = 509) taste loss. The frequency of reported loss of smell and taste in participants with and without SARS-CoV-2 antibodies are presented in n = 307) of those with complete smell loss had a positive test result. A total of 77.8% of participants with taste loss and 86.0% of those with complete taste loss (n = 209) had a positive test result. Extracts from questionnaire responses from participants with positive SARS-CoV-2 antibodies describing their loss of smell and taste can be seen in Among the participants who underwent SARS-CoV-2 antibody testing , and participants with combined loss of smell and taste were 4 times more likely to have SARS-CoV-2 antibodies . These findings remained unchanged after adjusting for sex, age, ethnicity, and smoking status.Participants with loss of smell alone were nearly 3 times more likely than participants with isolated taste loss to have SARS-CoV-2 antibodies  of the study participants reported neither cough nor fever. In our study cohort, loss of smell was more prevalent in participants with SARS-CoV-2 antibodies compared with those without antibodies , whereas taste loss was equally prevalent . Furthermore, participants with acute smell loss were 3 times more to be seropositive for SARS-CoV-2  compared with those with taste loss.In this community-based cohort study, undertaken during the peak of the COVID-19 outbreak in London, the seroprevalence of SARS-CoV-2 antibodies in participants with new onset loss of sense of smell and/or taste, was 77.6%. A total of 39.8% ; thus, it is plausible that the loss of taste reported by participants who also report loss of smell reflects impaired retronasal olfaction and hence, represents loss of flavor perception, as a consequence of smell loss. Interestingly, 6.6Globally, as populations are released from lockdown, early identification by the public of COVID-19 symptoms and rapid self-isolation and testing will be of vital importance to limit disease spread. Currently, as highlighted in SARS-CoV-2 specific antibody testing performed via telemedicine consultation enabled us to confirm participants’ reported symptomatology and verify their identity and test results, which adds to the quality of our data. Although viral nucleic acid detection using real-time PCR remains the gold standard for diagnosis, there are several limitations to this method, including a high processing time, labor intensity, and a high false negative rate –22. FurtThe main limitation of our study is the lack of a general population control group without loss of smell and/or taste. The study only recruited participants who reported acute smell and/or taste loss. Although this enabled us to study this presentation and its relevance to COVID-19, this also presents a degree of selection bias; hence, our findings refer to a population subset with new acute loss of smell and/or taste. In addition, although we used a questionnaire to assess COVID-19 symptoms and subsequently validated participants’ responses during the telemedicine interview, we did not undertake any objective assessments of smell or taste. However, from a COVID-19 disease containment perspective, the general public are unlikely to have rapid access to formal objective smell/taste testing. Moreover, our data show a high seroprevalence of SARS-CoV-2 antibodies in people who recognized a loss of their sense of smell. Furthermore, as our study relied on antibody testing via telemedicine, we were unable to account for COVID-19 patients who may not have seroconverted to develop IgG or IgM antibodies, which may lead to our findings underestimating the prevalence of COVID-19 in this population. However, our findings suggest that a key public health message is that people who notice a loss in their ability to smell everyday house-hold odors such as garlic, onions, coffee, and perfumes should self-isolate and seek PCR testing.The majority of our participants were female; this finding may reflect previous findings that females have a higher frequency of loss of smell and/or taste with COVID-19 than males ,9,28. WeIn a community-based population, the vast majority of participants with new onset loss of smell were seropositive for SARS-CoV-2 antibodies. Acute loss of sense of smell needs to be considered globally as a criterion for self-isolation, testing, and contact tracing in order to contain the spread of COVID-19.S1 STROBE checklist(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Healthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals. In this study we sought to determine if tracking smell sensitivity and loss using an at-home assessment could identify SARS-CoV-2 infection in HCW.P < .01). 6/9 (67%) of SARS-CoV-2-positive HCW reporting smell loss reported smell loss prior to having a positive SARS-CoV-2 test, and smell loss was reported a median of two days before testing positive. Neurological symptoms were reported more frequently among SARS-CoV-2-positive HCW who reported smell loss compared to those without smell loss .We performed a prospective cohort study tracking 473 HCW across three months to determine if smell loss could predict SARS-CoV-2 infection in this high-risk group. HCW subjects completed a longitudinal, behavioral at-home assessment of olfaction with household items, as well as detailed symptom surveys that included a parosmia screening questionnaire, and real-time quantitative polymerase chain reaction testing to identify SARS-CoV-2 infection. Our main measures were the prevalence of smell loss in SARS-CoV-2-positive HCW versus SARS-CoV-2-negative HCW, and timing of smell loss relative to SARS-CoV-2 test positivity. SARS-CoV-2 was identified in 17 (3.6%) of 473 HCW. HCW with SARS-CoV-2 infection were more likely to report smell loss than SARS-CoV-2-negative HCW on both the at-home assessment and the screening questionnaire (9/17, 53% vs 105/456, 23%, In this prospective study of HCW, self-reported changes in smell using two different measures were predictive of SARS-CoV-2 infection. Smell loss frequently preceded a positive test and was associated with neurological symptoms. It is increasingly clear that asymptomatic and pre-symptomatic infection play an important role in the ongoing spread of COVID-19 , 3. HealOlfactory impairment is a common manifestation of COVID-19, with meta-analyses indicating a prevalence of 77–86% in studies using standardized olfactory testing . Among pS1 Table), to track ratings of odor intensity perception using two household olfactory stimuli. High risk HCW undergoing routine (every 3 days) viral screening for SARS-CoV-2 infection were assessed for self-reported smell loss, along with symptoms commonly used to screen for COVID-19.The aim of this study was to determine if tracking smell sensitivity and loss using an at-home assessment could identify HCW who are infected with SARS-CoV-2. Since testing for objective smell loss using standard laboratory or clinical techniques is not feasible for widespread testing, we employed a brief at-home smell sensitivity screen, the Yale Jiffy  (IMPACT) study at Yale University . The goal of the parent study was to prospectively follow SARS-CoV-2-negative HCW at high risk of acquiring infection due to occupational exposures. The study recruited HCW working in the medical ICU or dedicated COVID-19 units at Yale New Haven Hospital (YNHH), a 1,541-bed tertiary care hospital located in New Haven, CT, USA. All participants provided written and/or verbal informed consent. Inclusion criteria for the IMPACT study included: a) aged 18 or older; b) English-speaking; c) working in a health care facility ; d) possible moderate to high risk exposure to COVID-19, or work in a COVID-19 unit, and e) SARS-CoV-2 negative at study entry. For this analysis of smell alterations and COVID-19, we excluded subjects without at least one SARS-CoV-2 PCR result and those who had not completed at least one daily symptom questionnaire or Yale Jiffy. All reported data were collected between March 31 and July 7, 2020.SARS-CoV-2 real-time quantitative polymerase chain reaction (RT-qPCR) testing was performed on self-collected nasopharyngeal and saliva specimens every three days. Specimens were processed and tested on the same day as collection following previously described protocols , 17. TwoThe Yale Jiffy is an online survey developed to screen for smell loss that can be conducted in under two minutes using readily available household items. The questionnaire includes two sections using self-ratings of: 1) ability to smell, and 2) strength of smell in response to olfactory and trigeminal stimuli. Peanut butter (or jam/jelly) was used as the olfactory stimulus as it has minimal or no trigeminal component, allowing for isolation of effects on the olfactory system. We also included a stimulus with a trigeminal component as a control stimulus .First, participants were asked to rate their ability to smell on a categorical scale (Poor/Average/Good/Very Good), to report any reduction in smell in the past week on a categorical scale (None/Slight/Moderate/Severe), and to rate the degree of reduction on a 10cm (0.0–10.0) visual analog scale (VAS). Next, participants were asked to hold the olfactory stimulus one inch from their nose and to provide ratings of both the strength of smell (0.0–10.0) and how different it smells from normal (0.0–10.0). HCW then held the trigeminal stimulus one inch from their nose and rated strength of sensation of irritation and difference from normal, as above. Since olfactory sensitivity fluctuates across the day , HCW werS2 Table), indicating how often they were bothered by common complaints caused by smell distortions, with responses ranging from “always” (1 point) to “never” (4 points). Parosmia was defined as a cumulative score less than or equal to 14 (out of 16). HCW responded to four hypogeusia screening questions for saltiness, sourness, sweetness, and bitterness, by indicating that they could detect these tastes “Easily” (3 points), “Somewhat” (2 points), or “Not at all” (1 point). Hypogeusia was defined as any total score less than the maximum of 12 points.As part of the IMPACT study, HCW completed an online daily symptom questionnaire. HCW were given a list of symptoms and prompted to indicate whether they had developed such symptoms in the past 24 hours. Listed symptoms included objective fever (>100.4°F), subjective fever, cough, shortness of breath, stuffy nose, sore throat, chills, sweating, malaise, fatigue, muscle pain, anorexia, nausea, vomiting, diarrhea, abdominal pain, and dizziness. In addition, we included screening questions for parosmia–changes in odor quality perception –and for Descriptive statistics were used to characterize the study population. The Fisher’s exact and Wilcoxon rank-sum tests were used to compare SARS-CoV-2-positive and negative HCW. Multivariable logistic regression models were developed to calculate adjusted odds ratios for the associations between smell symptoms and SARS-CoV-2 infection. Each model included one symptom as the predictor and additionally adjusted for age, sex, body mass index (BMI), ethnicity, and number of symptom surveys completed.To further examine the strength of association between symptoms of smell loss and SARS-CoV-2 infection, we compared HCW ever reporting smell loss to a subset of HCW never reporting smell loss who were well-matched on age, sex, ethnicity, and the number of daily symptom surveys completed. We used the R package ‘MatchIt’ for this analysis , specifyFinally, we summarized responses to the Yale Jiffy and daily symptom questionnaires. We also examined longitudinal responses among HCW who completed the questionnaires multiple times.P < .05 using R statistical software (version 3.4.2).All analyses were conducted with a two-sided statistical significance level of Fig 1). Within the sub-study population, 373 (79%) participants were female; the mean (SD) age was 37.5 (11.2) years; 375 (79%) were white non-Hispanic/Latino; 261 (55%) were registered nurses (RNs) and 98 (21%) were medical doctors (MDs) (Table 1). Compared to HCW included in this analysis, IMPACT HCW ineligible for the sub-study were more likely to be Black, Asian, or other ethnicity (P < .001) and have higher BMI (P = 0.004) (S3 Table).588 HCW were recruited and consented in the IMPACT study between March 31 and July 7, 2020, of whom 473 (80%) were eligible for the smell sub-study, while 115 (20%) were excluded because they did not undergo SARS-CoV-2 testing or they did not submit either a Yale Jiffy or symptom survey . Among the 313 (66%) HCW who completed the Yale Jiffy at least once, HCW completed a median of 10  Jiffy questionnaires.5771 SARS-CoV-2 RT-qPCR tests were performed on 473 HCW in the sub-study (median = 11 tests per HCW) between March 31 and July 7. Of these, 17 (3.6%) HCW tested positive for SARS-CoV-2.Table 1). Nine of the 17 (53%) SARS-CoV-2-positive HCW completed the Yale Jiffy at least once. SARS-CoV-2-positive HCW were more likely to report categorical smell loss on the Jiffy, with 5/9 (56%) SARS-CoV-2-positive HCW versus 43/304 (14%) SARS-CoV-2-negative HCW reporting smell loss  (Table 1). The five SARS-CoV-2-positive HCW who reported categorical smell loss via the Jiffy had a mean (SD) reduction in smell of 5.8 (4.0)cm on a 10cm (0.0 to 10.0) scale; those indicating severe loss had a mean 8.3 (3.0)cm decrease compared to 2.2 (0.5)cm in those with slight smell loss. For individuals with any smell loss reported via Jiffy, SARS-CoV-2-positive HCW reported more severe smell loss than SARS-CoV-2-negative HCW (P < .001) . SARS-CoV-2-positive HCW reported severe  or slight 2/5, 40%) smell loss on a categorical scale, while SARS-CoV-2- negative HCW reported slight  or moderate  smell loss.The demographic characteristics of SARS-CoV-2-positive HCW were similar to those of SARS-CoV-2-negative HCW (Table 1). Six of those seven also reported smell loss.All 17 SARS-CoV-2-positive HCW eligible for the smell sub-study completed at least one daily symptom questionnaire. Eight (47%) reported parosmia versus 83/456 (18%) SARS-CoV-2-negative HCW . Hypogeusia was reported in seven (41%) SARS-CoV-2-positive HCW and 104/456 (23%) SARS-CoV-2-negative HCW  . Of those who reported smell loss, 3/9 (33%) reported changes in smell as their first symptom; all 9 reported symptoms of COVID-19 in addition to changes in smell.Relative to Day 0 (defined as the day of test positivity), the median timing of reported smell loss was Day -2  among SARS-CoV-2-positive HCW reporting smell loss, with 6/9 (67%) reporting smell loss before test positivity .Overall, in either the daily symptom questionnaire or Yale Jiffy, nine (53%) SARS-CoV-2-positive HCW reported smell loss or parosmia, compared to 105/456 (23%) SARS-CoV-2-negative HCW . After adjusting for age, sex, BMI, and number of symptom questionnaires, smell loss remained a significant predictor of SARS-CoV-2 infection . Among other symptoms in the questionnaire, only dyspnea  and headache  were more strongly predictive of SARS-CoV-2 infection. In the matched analysis, unadjusted conditional logistic regression estimated a significant association between smell loss and SARS-CoV-2 infection  Table 2 summarizes findings among SARS-CoV-2-positive HCW stratified by smell loss as reported by either measure. Neurological symptoms as assessed by the daily symptom questionnaire, including headache, fatigue, and dizziness, were reported in all nine SARS-CoV-2-positive HCW reporting smell loss, and three (38%) who did not report smell loss. Prolonged neurological symptoms (> seven days after positive test) were reported in four (44%) SARS-CoV-2-positive HCW with smell loss versus one (13%) without smell loss. Three SARS-CoV-2-positive HCW, all of whom experienced smell loss, had significantly prolonged neurological symptoms (> 20 days after positive test).In this prospective study of a high-risk HCW population, we assessed smell loss alongside routine viral testing, finding that HCW who acquired SARS-CoV-2 infection over the course of the study had significantly increased odds of reporting smell loss compared to SARS-CoV-2-negative HCW. This finding was consistent across bivariate (OR = 3.7), regression-adjusted (OR = 4.5), and matched (OR = 6.0) analyses. Likewise, SARS-CoV-2-positive HCW reported significantly more severe smell loss compared to SARS-CoV-2-negative HCW. Overall, our findings add to the accumulating evidence demonstrating the efficacy and feasibility of using prospective, self-administered, at-home assessments of smell sensitivity to track changes over time in a group of individuals at high risk for SARS-CoV-2 infection.Our findings are consistent with recent research demonstrating robust associations between smell perturbations and SARS-CoV-2 infection. Specifically, smell and taste impairments are reported prior to (20%) or during 13%) hospitalization , typical% hospitaInterestingly, we found an association between reduction in smell ability and neurological symptoms in HCW with SARS-CoV-2 infection. While the exact pathophysiology underlying COVID-19 related smell loss remains incompletely understood, the association between smell loss and other neurological symptoms suggests a potential common mechanism underlying these symptoms. A growing body of evidence has demonstrated the potential for SARS-CoV-2 to infect neurons , 27, andOur study has several limitations. Despite performing almost 6,000 SARS-CoV-2 tests on nearly 500 HCW during the first peak of the COVID-19 pandemic in Connecticut, only 17 (3.6%) HCW tested positive, limiting the statistical power of our analysis. Nevertheless, we found large, significant effects. Smell sensitivity and loss using the Yale Jiffy were evaluated using both categorical responses and a VAS . Indeed,In our study, self-reported changes in smell perception were predictive of SARS-CoV-2 infection in a healthcare worker population. At-home smell assessments should be considered for non-invasive screening of groups that are at high risk for COVID-19.S1 FigResults of matching on age, sex, profession, ethnicity, and number of symptom questionnaires completed for HCW reporting smell on either survey , the daily symptom survey only , and the Yale Jiffy only . A, C, and E show the change an absolute standardized difference in means between the unmatched (“All Data”) and matched datasets. B, D, and F compare the distributions, as histograms, of the propensity scores between HCW with smell loss (“Treated”) and those without (“Control”) both before (“Raw”) and after (“Matched”) matching. In all cases, matching resulted in a decrease in the absolute standardized difference for the overall distance measure and a more similar distribution (as shown by histograms) of propensity scores.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableLaryngoscope. 2010;120(8):1708.Adapted from Landis BN, Frasnelli J, Croy I, Hummel T. Evaluating the clinical usefulness of structured questions in parosmia assessment. (DOCX)Click here for additional data file.S3 Tablea Unadjusted P values are Wilcoxon rank sum test (continuous variables) or Fisher’s exact test .Data are presented as median (IQR) for continuous variables and no. (%) for categorical variables. Abbreviations: BMI, body mass index; HCW, healthcare workers; IMPACT, Implementing Medical and Public Health Action against Coronavirus (CT); MD, medical doctor; RN, registered nurse. (DOCX)Click here for additional data file.S4 Tablea Odds ratios are presented for the association between smell loss and SARS-CoV-2 infection as calculated from unadjusted conditional logistic regression after matching on demographics and symptom questionnaire participation.Abbreviations: CI, confidence interval. OR, odds ratio. (DOCX)Click here for additional data file.


Some nonlinear radiations such as superfluorescence can be understood as cooperative effects between atoms. We regard cooperative radiations as a manifested effect secondary to the intrinsic synchronization among the two-level atoms and propose the entanglement measure, concurrence, as a time-resolved measure of synchronization. Modeled on two cavity-coupled qubits, the evolved concurrence monotonically increases to a saturated level. The finite duration required for the rising to saturation coincides with the time delay characteristic to the initiation of superfluorescence, showing the role of synchronization in establishing the cooperation among the qubits. We verify concurrence to be a good measure of synchronization by comparing it with asynchronicity computed from the difference between the density matrices of the qubits. We find that the feature of time delay agrees in both measures and is determined by the coupling regimes of the cavity-qubit interaction. Specifically, synchronization is impossible in the weak coupling regime. How synchronizations arise in different situations has since remained a problem of interest2. In recent years, the study of the classical phenomenon has been revived under the quantum regime. Synchronization is observed between a pair of nanomechanical oscillators3 and on the motions of the Cooper pairs among Josephson-insulated superconducting islands4. It is also ubiquitously predicted between a qubit and an oscillator5, between a cavity field and an oscillator6, between two oscillators7, among a trapped group of cold atoms8, and even between a quantum Van der Pol oscillator and an external drive9.Centuries ago, Huygens studied the correlation among the motions of pendulums and discovered the synchronization pattern of these individual oscillators under the influence of a common oscillator they are coupled to11. This nonlinear fluorescent effect embodies Dicke’s formulation of superradiance12, where the nonlinearity exemplifies in the N atoms. Furthermore, the emitted photonic pulse peaks after a positive time delay14 that bears no direct dependence on the atom-field coupling strength, showing the collective interatomic motion to be a nonlinear process. Experimentally recorded on hydrofluoric gas15, cesium16, and most recently rubidium vapor17, this delay shows the necessity of a finite time during which the atoms establish cooperation before the radiation is initiated18.Here we study the synchronization of two qubits coupled indirectly to each other through a cavity field, in specific relevance to the quantum optical phenomenon of superfluorescence21 of two cavity-coupled qubits, which is the minimum-dimensional system that emulates the structure of an atomic ensemble undergoing cooperated radiation.Such a delay is also characteristic of synchronization: it takes finite time for the classical Huygens pendulums to reach in-phase oscillations. If one regards the correlated atoms as quantized Huygens oscillators concentrated on the lowest two levels at the weak-energy limit, it is not hard to find the resemblance between cooperated radiation and synchronization. In this paper, we formalize the study of this resemblance by analyzing the entanglement dynamics22, quantum dots23, and resonator modes24. We note that synchronization as a quantum correlation can be studied using quantum discord25, as it was for two oscillators26, and non-locality. The advantage of entanglement approach is avoiding the unnecessary constraint to two-party correlation. We use specifically, in contrast to quantum discord, concurrence27 as the entanglement measure to quantify quantum synchronization in order to extract the temporal features of atomic cooperation. Concurrence is broadly applicable, especially for multipartite system. After it was introduced for two-qubit systems27, it was soon extended to the cases of three qubits28 and N-qubits29. Concurrence has then been generalized to two multi-level systems31 and finally to multipartite multi-level systems32. Given its generality, concurrence permits synchronization to be exemplified not only between two qubits, but among three parties that include the cavity field. In addition, synchronization is present in a Hilbert space of finite dimensions33, so non-locality is not an essential factor to initiate synchronization.Many different metrics have been proposed over the years to measure synchronization of different quantum systems, such as a set of two-level systems in a shared bath35. Therefore, we extend the employment of multipartite concurrence to the dynamical analysis of the finite-dimensional cavity-qubit system. It is found that the entanglement among the components universally begins at a zero level and rises to a saturated value after a finite delay. The length of the delay and the saturated level depend nonlinearly on the cavity-qubit coupling strength. We use numerical simulations to show that the nonlinear dependence is divisible into weak, strong, and ultrastrong coupling regimes, verifying the role of operation regimes36 in the dynamics of superconducting circuits. Under all coupling strengths, the saturated entangled state is sustained thereafter, where oscillations in concurrence only exists locally about the saturation offset with an amplitude much smaller than the offset. These distinctive features of a synchronized state stand in constrast to the sudden death or death and revival37 one usually sees in entanglement evolution.Further, concurrence is proved to be a good measure for static analysis of collective phenomena such as radiationx, p)-quadratures of quantum oscillators24. The transition point in time produced from the synchronization measures matches exactly the delay time found in the concurrence evolution, proving that the qubit-to-qubit synchronization is well registered in the entanglement. Moreover, since two-qubit systems on a superconducting circuit can produce superfluorescent pulses38, the synchronization delay is associated with the initiation of superradiance, thereby establishing the dynamic correlation between entanglement and the atomic cooperation for collective phenomena.To verify the coincidence between the maximization of concurrence and the dynamic process of synchronization, we introduce a synchronization measure computed from the density matrix, which is modified from the synchronization measure introduced on the  system where each qubit is coupled to the cavity through dipole-field interaction under the rotating wave approximation. The parameters adopted for the numerical analysis are sourced from the superconducting circuit implementationThe total system Hamiltonian A and B when writing the product states and let the first letter denote the state of the left qubit, the middle letter that of the cavity mode, and the last letter that of the right qubit , B (the right qubit), or C (the cavity), i.e. The permitted dressed level transitions induced by the external driving can be found by substituting Eq.  into Eq.A with j and k being the row and the column indices, respectively. We denote A is integrable, then solving the linear system for In the rotating frame s in Eq. , one canTo see that the evolution of the state vector can initiate the synchronization of the two delocalized qubits, we assume the cavity mode is initially driven by the external field to reach a partial population inversion while setting the qubits initially at the ground. In other words, the expansion coefficients at the initial moment are: 40: 42.We plot out the evolutions of these coefficients in Fig. entclass1pt{minimaentclass1pt{minimaWe observe that for both the lower set and the upper set of states, there exists a transition point of the oscillations of the coefficients, which is located at about To fully capture the evolution characteristics of the two cavity-coupled qubits from a holistic point of view, we apply two entanglement measures—bipartite concurrence and tripartite concurrence—to the state vector of the total system.The bipartite concurrence quantifies the inseparability of the joint pure state of two coupled systems of arbitrary dimensions by inverting the density matrix. For our case here, the joint state is the product state Applying ⟩ in Eq.  to the fentclass1pt{minima28. Extending the original formulation on three-qubit systems, we generalize the inversion operations for two arbitrary-dimensional systems given above to three arbitrary-dimensional system. That is, we introduce the superoperatorI denotes the identity matrix while The cavity mode plays an active part in initiating the entanglement between the two qubits. From the entanglement-theoretic point of view, the concurrence is distributed among the qubits as well as the cavity. Taking away the pairwise entanglements between any two parties in the tripartite system, one obtains the residual concurrence that remains as an equally distributed entanglement among all three partiessing Eq. , we plotComparing Figs. The concurrences plotted in Fig. 44 and even up to hundreds of µs when an error-correcting feedback mechanism is implemented45.We note that the dynamics originated from the equations of motion –18) doe doe18) dThe synchronization between the qubits is also affected by how strong they are driven by the cavity mode, i.e. the coupling strengths Multi-partite concurrence as a measure of synchronization reveals a gradual increase between two qubits in the time domain, explaining the existence of a delay in the superfluorescent pulse of cooperated radiation from a system-intrinsic point of view. This synchronization is affected by many factors, among which the symmetry of the system parameters plays an important part. Tuning the system from a symmetric setting to an asymmetric setting is accompanied by tuning the transition rates of the qubits from a synchronous setting to an asynchronous setting. For the latter, we refer to the scenario where the population of the left qubit oscillates at a Rabi frequency not synchronous to that of the right qubit.24 for continuous variable systems to discrete systems. We consider instead the measure of asynchronicityHowever, since two oscillators sharing a common oscillating platform are able to synchronize after certain time duration according to classical mechanics, we expect the qubits sharing the cavity resonator would behave similarly. To precisely describe the transition process from asynchronous regime to synchronous regime, we extend the quantum synchronization measure introduced in Ref.46. For example, a 43. To consider the scenario where the sychronization is initiated from the cavity field as the medium, we let the initial state be only populated in the first and second excited state of the cavity mode, i.e. No matter the coupling strength, there exists a transition point after which the asynchronicity remains at a stable value. This transition point is identical to the transition point shown in Fig. Before reaching the stable minimal value, the asynchronicity increases from a non-zero value for a certain duration, which are spent on the cooperation by the qubits. When the coupling is sufficiently weak  for for\docu