The composite was applied as a glassy carbon electrode modifier (GCE) to enhance dopamine (DA) and uric acid (UA) detection. The ternary PFSG composite was compared with its binary precursor Fe2O3-SnO2/reduced graphene oxide (FSG). The influence of the modified GCE electrodes on their performance as a sensing platform was determined. GCE/PFSG showed better sensing parameters than GCE/FSG due to the introduction of polyaniline (PANI), increasing the electrocatalytic properties of the electrode towards the detected analytes. GCE/PFSG enabled the detection of low concentrations of DA (0.076 µM) and UA (1.6 µM). The peak potential separation between DA and UA was very good (180 mV). Moreover, the DA oxidation peak was unaffected even if the concentration of UA was ten times higher. The fabricated sensor showed excellent performance in the simultaneous detection with DA and UA limits of detection: LODDA = 0.15 µM and LODUA = 6.4 µM, and outstanding long-term stability towards DA and UA, holding 100% and 90% of their initial signals respectively, after one month of use.A ternary polyaniline/Fe In recent years, electrochemical detection has been one of the most widely used techniques for analyte detection, mainly due to its simplicity, fast response and sensitivity . CurrentGraphene-based materials, due to their unique structure, primarily improve the conductivity of composites applied in electrochemical measurement systems ,19,20. O2O3, SnO2, Fe3O4, ZnO, NiO and CuxO [2) have been widely reported, indicating its suitability for sensing applications [2 can also be considered a catalyst support [2 as a carrier of iron oxide (Fe2O3), which is a low-cost and stable catalyst [A wide range of transition metal oxides has also been investigated for use in electrochemical detection systems, including Feand CuxO ,26. The and CuxO . The catications ,31,32. S support . The abo2 nanocrystals and graphene oxide (GO) using AA to obtain reduced graphene oxide (rGO)-SnO2 and then applied the as-prepared composite to detect DA. Due to the large number of active sites on the nanocomposite surface, the sensor showed good electrochemical performance, presenting a very low limit of detection (LOD) for DA (6 nM). Despite the low LOD, the proposed sensor worked only in a narrow concentration range (from 0.08 to 30 µM). Another binary graphene-SnO2 nanocomposite was proposed by Sun et al. [2 nanoparticles, from a hydrothermal synthesis, were mixed with graphene and ultrasonicated. The LOD for DA was 130 nM. In the interference studies, the peak-to-peak separation between DA and UA was 170 mV, indicating no interference of UA with DA. In the detection of UA, a platform based on a hydrothermally synthesized α-Fe2O3/polyaniline nanotube (PAn NTs) composite was applied [3O4-SnO2-Gr nanocomposite as an electrochemical platform for the simultaneous detection of DA, UA and AA. A thermal exfoliation of graphite was used to prepare graphene nanosheets that were mixed with SnO2 and Fe3O4 precursors and sonicated to obtain Fe3O4-SnO2-Gr. The ternary composite exhibited better electrochemical performance in regard to the simultaneous detection of DA, UA and AA than the graphene-modified electrode. The obtained LODs for DA, UA and AA were 7.1, 5.0 and 62 nM, respectively. However, this sensor operated at pH 5.5, which, from the point of view of real sample analysis, requires an additional pH adjustment step. All the aforementioned studies show that multicomponent platforms for DA and UA detection require an appropriate design to overcome issues with sensor sensitivity, selectivity and stability. Binary nanocomposites are very often applied in electrochemical sensing due to their unique properties, but currently, there is a growing interest in using ternary nanocomposites based on CPs, which play a significant role in the detection of various analytes [Due to their multifunctionality, nanocomposites significantly improve the working parameters of sensors in comparison to their pure counterparts, which has been thoroughly reported recently . To daten et al. . SnO2 na applied . The aut applied . Therefoanalytes .2O3-SnO2/rGO nanocomposite (PFSG) for DA and UA detection. We compared ternary nanocomposite performance with its binary precursor Fe2O3-SnO2/rGO (FSG). Despite the complexity of the proposed composites, we applied a simple hydrothermal synthesis route. A beneficial impact on DA detection was observed after the introduction of PANI to the composite structure. The LODs for DA were 1.5 and 0.076 µM for the binary and ternary nanocomposites, respectively. The working pH of the sensors for DA and UA detection was 7.0 and 6.6 respectively, which demonstrates the great applicability of the sensors in real sample analysis due to the pH value being close to the physiological pH of humans. Moreover, the detection of DA was possible even if the concentration of interfering UA was ten times higher. The application of PFSG allowed us to obtain excellent peak-to-peak separation between DA and UA (180 mV). The modified glassy carbon electrode (GCE)/PFSG presents outstanding long-term stability in DA detection, maintaining ~100% of its initial signal after one month of use.In this study, we propose a promising platform based on the ternary PANI/Fe2O3 (JCPDS card no. 33-0664) and tetragonal SnO2 (JCPDS card no. 41-1445) [2O3 at approximately 24° and the SnO2 peak at approximately 26.5° (110) overlap with the less intense diffraction peak corresponding to the (002) plane of the rGO graphitic structure [The XRD measurement results of the binary and ternary composites are presented in 41-1445) ,36. The tructure . After ttructure .2O3 and SnO2 nanoparticles may act as spacers during the hydrothermal reduction of GO, preventing the strong aggregation tendency of graphene sheets [2O3 takes on a cubic form with dimensions up to 200 nm in size. PANI partially conceals the iron oxide nanocubes, leading to the formation of the conductive polymer layers on the inorganic component. The smallest nanoparticles on the surface of the rGO are SnO2, which maintain their structure compared with their structure in the binary composite.e sheets . The secBET), the total pore volume (Vt) and the mesopore volume (Vmes), are presented in 0 -specific surface area  [First, the electrochemical performance of the bare GCE, GCE/FSG and GCE/PFSG electrodes towards DA and UA was evaluated using CV . The bar-quinone   . Moreove [2 g−1) ,48.a = 5.6) under these conditions [−1) on  on  GCE/FSG  [−1) on  GCE/PFSG [−1) on  100 µM D [−1) on  300 µM UDA = 1–35 µM, LRUA = 5–40 µM; GCE/PFSG: LRDA = 0.1–20 µM, LRUA = 5–50 µM), and the second range includes high analyte concentrations . Both the low and high linear working ranges overlap, which enable the detection of DA and UA over a wide concentration range with high accuracy. The correlation coefficients of the calibration plots were in the range of 0.989–0.997, indicating suitable selection of the linear working scope. The upper limits of the LRs for DA detection with both modified electrodes are comparable. However, a clear difference is observed in the case of detection in the range of low concentrations. The GCE/PFSG electrode enables the detection of DA even at nanomolar concentrations. The lower limit of the LR for GCE/PFSG is ten times lower than that obtained for GCE/FSG (0.1 vs. 1 µM). This outstanding behavior can be explained by the appropriate amount of electronegative oxygen groups in the structure of the ternary composite in comparison to the binary composite [S is the standard deviation of the blank sample and b is the slope of the calibration plot. A great improvement in LOD is observed for the electrode modified with the PFSG nanocomposite in comparison to that of GCE/FSG . The sensitivity of the sensor is also significantly improved from 2.04 to 2.75 µA µM−1 for GCE/FSG and GCE/PFSG, respectively. The better performance of the GCE/PFSG-based sensor can be ascribed to the chemical composition of PFSG, which contains an optimal oxygen content in the composite, and the presence of conductive PANI in the structure of the material, which provides improved electrical conductivity. In the case of UA detection, the LOD value and the sensitivity are comparable for both electrodes: 1.8 µM and 1.2 µA µM−1 for GCE/FSG and 1.6 µM and 1.1 µA µM−1 for GCE/PFSG, respectively. Moreover, the potential of DA oxidation is reduced from 360 to 320 mV due to the presence of PANI. In the case of UA oxidation, the peak potential shifts towards negative values from 500 to 470 mV for GCE/FSG and GCE/PFSG, respectively. As a result, the DA-UA peak potential separation is 140 and 150 mV for GCE/FSG and GCE/PFSG, respectively. The calibration plots of DA and UA oxidation on GCE/FSG and GCE/PFSG were performed by DPV , which is a suitable technique to evaluate faradaic processes . Figure omposite  and by tomposite . The rolomposite ,55. The omposite ,49. The 2O3-SnO2) for analyte molecules, which plays a catalyzing role in the redox reaction [The decrease in the oxidation potential of both molecules on GCE/PFSG may result from the separation of FSG sheets by long polymer chains embedded between the composite sheets . This sereaction ,31,32,33reaction  proposed−1). In contrast, a large decrease in the sensitivity, up to 25% of the initial value (1.1 vs. 0.27 µA µM−1), is observed in UA detection. This result may indicate that the active sites present on the electrode surface are mainly occupied by DA molecules.Due to the better DA and UA sensing performance of the DA and UA for the GCE/PFSG electrode compared with GCE/FSG, further studies were carried out with an electrochemical detection set and this electrode. To investigate the selectivity of the GCE/PFSG-based sensor towards the detection of DA and UA, DPV measurements were performed when the concentration of one analyte was kept constant . The lowTo examine selectivity more accurately, the simultaneous detection of DA and UA was also investigated. 2O3-SnO2 phase and PANI. The graphene sheets provide nucleation sites for PANI during the hydrothermal treatment and can be treated as a scaffold for the polymeric chains, which enhances the mechanical strength of the ternary nanocomposite [The reproducibility of the GCE/PFSG sensor was evaluated by preparing five different modified electrodes and comparing their anodic responses towards DA and UA. The relative standard deviation (RSD) of the DA and UA oxidation signals are 3.2% and 5.1%, respectively. The repeatability was determined by recording the oxidation signal of five independent solutions of 100 µM DA and 300 µM UA. The obtained RSD values are 3.3% and 3.4% for DA and UA, respectively. The low RSD values indicate that the fouling effect at the electrode surface induced by the oxidized forms of DA and UA is minimized , leadingomposite ,54.To evaluate the suitability of the designed sensor in real sample analysis, standard addition method was applied for three artificial urine samples. The artificial urine was diluted 10 times in 0.1 M PBS, and subsequently certain amounts of DA and UA were added into the sample. The amount of the analyte was estimated by comparing the peak current with the calibration curves . The resThe GO synthesis procedure that uses a modified Hummers method is included in the PANI was prepared using the oxidative polymerization method previously described . Briefly3)3 and SnSO4 . First, 135 mg of aqueous GO solution was mixed with 0.3 mmol Fe(NO3)3 and 0.15 mmol SnSO4 and ultrasonicated for 1 h. Next, 1 mL of 10% v/v ammonia solution was added to the mixture to establish a pH value of 9. The prepared suspension was then placed in a stainless-steel autoclave . The reaction was performed at 180 °C for 8 h under constant stirring at 200 rpm. The resulting product was washed with Milli-Q water and subsequently vacuum-dried overnight at 60 °C. The expected mass ratio of mixed Fe2O3-SnO2 to rGO in the composite was 1:2.FSG was obtained using a hydrothermal treatment of GO in the presence of Fe and 10 µM of Nafion. Then, the as-prepared dispersions were ultrasonicated for 3 h to obtain a homogenous mixture. Alumina oxide slurries (granulation: 0.3 and 0.05 µm) were used to polish the GCE. After polishing, the electrodes were washed with water. Finally, the cleaned GCEs were modified by drop casting 2.5 µL of the FSG and PFSG dispersions and dried under an infrared lamp.The dispersions that were used to modify the GCE were prepared by mixing 4 mg of FSG or PFSG with 1 mL of dimethylformamide , Milli-Q water . The quenched solid density functional theory (QSDFT) was used to determine the pore size distribution based on the nitrogen adsorption data.The morphology of the prepared composites was examined using field emission scanning electron microscopy (FESEM) with a Merlin Zeiss Instrument . The chemical surface composition and distribution of functional groups were analyzed by X-ray photoelectron spectroscopy (XPS) using a PHI 5000 VersaProbe Instrument . X-ray diffraction (XRD) patterns were measured using an Ultima IV Rigaku diffractometer with a Cu Kα radiation source (λ = 1.54056 Å). The porous structure of the composites was determined by Nscan) of 100 mV/s. The calibration plots of DA and UA were obtained by differential pulse voltammetry (DPV) with optimized parameters, including pulse amplitude, pulse width, pulse period and pulse increase . A graphite rod, Ag/AgCl/3.5 M KCl electrode and GCE/FSG or GCE/PFSG electrode were used as the counter, reference and working electrode, respectively. All electrochemical measurements were performed in a phosphate-buffered saline  solution at an appropriate pH. Cyclic voltammetry (CV) measurements were performed in a potential range from −0.5 to 0.8 V (vs. Ag/AgCl/3.5 M KCl) at a scan rate , the introduction of PANI decreased the oxidation potential of both molecules due to the increased access to catalytic sites in the entire structure of the material, thereby resulting in the good resolution of the DA and UA oxidation peaks. Both fabricated sensors showed very good performance in DA and UA detection. The GCE modification with PFSG provided wider linear concentration ranges for DA and UA detection  in comparison to that of GCE/FSG  and a lower LOD for DA (0.076 µM for GCE/PFSG and 1.5 µM for GCE/FSG). The GCE/FSG and GCE/PFSG sensors enabled the detection of UA with a LOD of 1.8 and 1.6 µM, respectively. The application of GCE/PFSG for simultaneous DA/UA detection in the presence of interfering compounds resulted in good peak separation while maintaining the intensity of the DA oxidation peak, which was almost unaffected. These results clearly indicated the outstanding selectivity of the fabricated sensor towards DA. Moreover, the excellent long-term performance of GCE/PFSG confirmed its potential as a sensing platform, exhibiting 100% of the initial DA signal after 30 days. Additionally, the real sample analysis revealed good applicability of the GCE/PFSG electrode in detection of DA and UA.In this paper, we proposed a highly promising polyaniline/Fe


Fertility knowledge is vital to the fertility health of young people and greatly impacts their fertility choices. Delayed childbearing has been increasing in high-income countries, accompanied by the risk of involuntary childlessness or having fewer children than desired. The aim of this study was to investigate knowledge about fertility issues, the related influencing factors, the method of acquiring fertility knowledge, and the relationship between fertility knowledge and fertility intentions among college students.An online cross-sectional survey of Chinese college students was conducted in Hunan Province from March to April 2018. A total of 867 college students from three comprehensive universities responded to a poster invitation utilizing the Chinese version of the Cardiff Fertility Knowledge Scale (CFKS-C). Data were explored and analysed by SPSS (version 22.0) software. Descriptive statistics, chi-squared tests, T-tests, and Pearson’s correlations were used for the measurements.p<0.05). A total of 81.9% of the participants reported that they would like to have children, the average score of the importance of childbearing was 6.3 (SD = 2.7), and the female score was lower than the male score (p = 0.001). A small positive relationship was observed between the CFKS-C and the importance of childbearing . Respondents indicated that they gained most of their knowledge from the media and internet (41.4%) and from schools (38.2%).The average percent-correct score on the CFKS-C was 49.9% (SD = 20.8), with greater knowledge significantly related to living in a city district, being not single status, majoring in medicine, being in year 4 or above of study, and intention to have children (all Yong people in college have a modest level of fertility knowledge, a relatively low intention to have a child, and deficiencies in fertility health education. There is a need to improve the accessibility of fertility health services by developing a scientific and reliable fertility health promotion strategy. Fertility is one of the major components of population growth and age structure change and is central to the Programme of Action of the International Conference on Population and Development. The past six decades have witnessed a major decline in world fertility, especially in the developing world , 2. The Reasons for low fertility are multifactorial and often difficult to disentangle. The published literature has stated that despite changes in social and economic development, the lack of knowledge about fertility and transformation of fertility concepts are crucial. An international survey of 79 countries from the International Fertility Decision-making Study (IFDMS) showed that the average score on a test of fertility knowledge was 56.9% . Other sIn prior studies, college students, as a particular group of young people, have been in focus given that they are of reproductive age and face choices between education and career versus marriage and childbearing. Previous surveys among young college students in Sweden, Italy, America, and New Zealand revealed that knowledge about human fertility was rather poor , 22–24. Therefore, the aim of the present study was to investigate the level of fertility knowledge among college students in Hunan Province, the related influencing factors, the methods by which students acquire fertility knowledge, and the relationship between fertility knowledge and fertility intention.A cross-sectional survey of college students was conducted in Hunan Province, China, during a one-month period of March 8 to April 8, 2018.The survey was conducted in Hunan Province, China, which is located in the middle of China. In this province, student enrolment in regular institutions of higher education is 22.1 persons per 10,000 population, and there are 109 schools that are regular institutions of higher education . Three cInclusion criteria: 1) Aged between 18 and 30 years old; 2) Full-time college students of regular institutions of higher education; 3) Informed consent and voluntary participation. Exclusion criteria: 1) Non-Chinese student; 2) Pregnant; 3) Have children. Students were recruited by open recruitment as advertised by poster. These posters, including a brief description of the study and a QR code, were posted on the notice boards of each university, canteen and dormitory in the university to be seen by all students and staff. By scanning the QR code using a mobile phone, students could obtain a brief description of the study, an informed consent form and the questionnaire (Questionnaire star software). The questionnaire star software , the first and largest domestic online questionnaire survey and test platform, has sent more than 24.6 million questionnaires and collected more than 1.6 billion questionnaires from respondents. The online survey was open from March 8 to April 8, 2018. Participants who completed the questionnaire were provided a reward incentive via the internet.n = uα/22π(1 − π)/δ2 Uα/2 = 1.96, δ = 0.05, π = 0.569 [The sample size was estimated by the formula  = 0.569 , put intThe questionnaire consisted of 25 items covering 4 domains. It was pilot tested twice with 30 college students to ensure that the instrument was understandable. The pilot data were not included in the analysis.Participants stated their age in years, gender, place of birth, whether they were of the only child generation (yes/no), sexual orientation, relationship status, their discipline, and the year of study.One question investigated the personal intention to have children (yes/no), and one 0- to 10-point response scale surveyed the personal perceived importance of childbearing .The Chinese version of the Cardiff Fertility Knowledge Scale (CFKS-C) was translated into Chinese from the original Cardiff Fertility Knowledge Scale (CFKS) developed by Boivin et al. (2013) and shown to have satisfactory validity and reliability. The CFKS consists of 13 items. Three areas of knowledge about fertility are measured: risks for reduced fertility, misconceptions about fertility, and basic facts about infertility. A three-point scale of “true”, “false”, or “do not know” was used to rate all items. One point was given for a correct answer, and zero points were given for an incorrect or unknown answer. Scores were reported as percentages that were equal to points divided by the total number of questions. In the original CFKS, the internal consistency coefficient alpha (Cronbach’s α) was 0.79 . The CFKIn addition, three questions were used to investigate students’ perceived knowledge of fertility issues: (I) their perceived education level of fertility-related knowledge , (II) whether or not they discussed fertility issues with family (yes/no), and (Ш) where they gained most of their knowledge about fertility issues .P value less than 0.05. All analyses were performed using SPSS (version 22.0) software .Descriptive statistics were used to describe the sociodemographic variables and fertility knowledge. Categorical data were compared using Chi-squared tests. T-tests and analysis of variance (ANOVA) were used to compare the total scores on the CFKS-C between sociodemographic categories. Pearson’s correlation was used to examine the association between the importance of childbearing and the CFKS-C score. The data were assessed for errors before double-entry computer input. Statistical significance was defined as a two-sided Ethical review and approval were performed by the IRB of behavioural and nursing research in the School of Nursing of Central South University (Project Number 2017028). Permission to use the CFKS was obtained from the authors who developed it. A cover letter was presented to respondents to explain the aim and process of the study before the questionnaire was shared. After participants read the contents of the consent form and agreed to participate in the study, they checked “consent” button and began to fill in the questionnaire. Participation in this study was voluntary, anonymous and confidential. To maintain anonymity, participants were asked not to provide their name or telephone number. Collection of online data complied with the “Code of Conduct of Marketing Research”. All data collected were treated anonymously and confidentially.The average age of respondents was 19.7 (SD = 2.0), ranging from 18 to 30 years old. Female participants accounted for 56.8% of the sample, and 68.8% of participants were single. The sociodemographic characteristics of the participants are summarized in Table The average percent correct score on the CFKS-C was 49.9% SD = 20.8). Table 0.8. TablAs shown in Table p = 0.039). When participants were asked to rate the importance of childbearing on a scale of 0–10, the average score was 6.3 (SD = 2.7). Female’s scores  were lower than male’s  . A small positive relationship was observed between CFKS-C and the importance of childbearing .Among participants, 81.9% said that they would like to have children, while 18.1% reported that they had not thought about it. The CFKS-C scores  of those who intended to have children were recorded, and these scores were significantly higher than the scores of those who did not intend to have children   of the participants reported that they were “somewhat educated” about fertility issues, followed by “educated” (30.9%) and “not educated at all” (10.4%). Only 4.5% of the participants stated that they were “highly educated”. Respondents stated that they gained most of their knowledge from the media and internet (41.4%), schools (38.2%), family (9.1%), friends (6.6%), doctors/gynaecologists (2.9%), and non-government organizations (1.5%).The current study found that young college students in China (mainland) had a modest level of fertility knowledge. This finding was consistent with the findings of previous studies , 13. TheThe findings of the present study also indicated that most of the participants had misconceptions about fertility and the basic facts of infertility. Table All participants with medicine discipline had increased fertility knowledge, as did those who were female, born in a city, those with non-single status, those being in year 4 or above of study, and those with an intention to have children. These findings are consistent with previous studies, which reported that greater fertility knowledge was associated with female gender , 13, 22,Approximately one-fifth (18.1%) of the respondents stated that they did not want to have children, which was higher than previous studies in the US (14.2%) and Sweden (4.1%) , 23 and Moreover, this study found a small positive relationship between CFKS-C and the importance of childbearing. Therefore, improving fertility knowledge may be beneficial for increasing the fertility rate.Overall, more than three-quarters of participants self-reported that they had some education about fertility-related knowledge, and more than half discussed fertility issues with family, but their rate of correct fertility knowledge was actually low. Furthermore, the findings showed that more than half of college students obtained knowledge from media and the internet (41.4%) instead of school and the health department. However, in the US, the main modes of acquiring fertility knowledge were school 46.0%) and family (20.0%) . These f.0% and fFirst, the cross-sectional design of the present study had self-selection bias, which was inherent in using the convenience sampling method.Second, the response rate and the characteristics of the non-respondents could not be ascertained because the participants were recruited by posters at the university.Third, the population in three universities in Hunan province may not reflect the fertility knowledge level in other universities of Hunan province or other parts of China when generalizing the findings.In addition, to collect more credible and comprehensive information, both quantitative and qualitative methods could be used in the future.This study reveals that college students have a modest level of fertility knowledge, a relatively low intention to have a child, and deficiencies in fertility health education. There is a need to improve the accessibility of fertility health services by developing a scientific and reliable fertility health promotion strategy. Because fertility decisions should be based on correct and complete information, educational institutions and providers need to take action to embed fertility education into curricula for college students and the general public.


Ligustri lucidum fruit samples.In this work, we reported a facile wet chemical method for depositing Pt nanoparticles on the surface of boron nitride nanosheets (BNNS-Pt NPs). The deposited nanocomposite was applied for glassy carbon electrode surface modification. The modified electrode was then used for detecting ursolic acid (UA). The results indicate that the BNNS-Pt NPs exhibited excellent electrocatalytic activity toward UA oxidation compared with that of the bare glassy carbon electrode (GCE) and Pt NPs/GCE. The UA oxidation currents is linearly related its concentration from 1 to 1,200 pM. The limit of detection can be calculated to be 0.5 pM. In addition, the UA sensor was also successfully used for the determination of UA in  Ligustrum lucidum Ait plant . UA is a pentacyclic triterpenoid identified in many herbs. UA has many biological effects, such as acting as a sedative, having anti-inflammatory, antibacterial, anti-diabetes, and anti-ulcer effects, and reducing blood sugar  was better than that of the control group with the same treatment time. Clinical trials prove that UA shows a significant and rapid reduction in alanine transaminase, elimination of jaundice and recovery of liver function. After 3 weeks of treatment, the negative rate of 21 cases of HBeAg-positive patients was 61.9%. The negative rate of 21 cases of HBsAg positive patients was 42.8%, indicating that it also has a certain therapeutic effect on hepatitis B. In addition, Ramos-Hryb also confirmed that UA not only has the effect of treating viral hepatitis but also acts as an antidepressant; it contains UA perilla extract as a treatment for depression  can treat rat liver toxicity  for the electrocatalytic determination of UA. Meanwhile, the proposed approach has been successfully used for determining UA content in the Ligustri lucidum fruit extract.In this study, we report a facial strategy for the The exfoliation of BN was performed according to the literature and contains two steps . After the pH was adjusted to 10 using 0.1 M NaOH, the solution was stirred for 24 h at room temperature. After centrifugation and washing twice with water, the resulting BNNS-Pt NP composite was obtained and re-dispersed in 10 mL water for further use.The BNNS-Pt NP composite was prepared by growing Pt NPs Scanning electron microscopy  was used for morphology characterization. XRD characterization was carried out with a BRUKER D8–Advance XRD system with Cu Kα radiation. All the electrochemical sensing and characterizations were conducted using a CHI 832 electrochemical workstation. All the experiments were performed at room temperature.Ligustri lucidum (fruit) were purchased from a local traditional Chinese medical center. For the real sample preparation, the dried Ligustri lucidum (fruit) was grounded and then dispersed into 50 mL methanol with vigorous stirring for 12 h. Then, the dispersion was filtered through a 0.45 μm filter paper and diluted to 50 mL by PBS.A glassy carbon electrode (GCE) was polished by 0.05 μm alumina dispersion and cleaned by water and ethanol. Then, 4 μL of catalyst dispersion (0.2 mg/mL) was dropped onto the GCE surface and dried naturally. The anti-interference study was carried out by square wave voltammetry (SWV) in the presence of UA and interference species. Triplicate measurements were recorded and the average values of current changes were calculated. Peanut shells and 6]3−/4− in 0.1 M KCl by electrochemical impedance spectroscopy (EIS). As shown in The electrochemical properties of bare GCE and modified GCE were investigated using 5 mM [Fe(CN)6]3−/4− redox is largely suppressed with a larger peak to peak splitting, indicating that the insulation of the BNNS lowers the electron transfer rate, which agrees with the EIS result , PBS (pH 7.0), and Tris (pH9.5). The PBS showed the best performance. Therefore, we selected the PBS in this work. pa(V)=(−0.0211) pH+0.0802 (R = 0.997). The number of electrons (n) in the overall reaction can be calculated as 2 for a totally irreversible diffusion-controlled process.The effect of pH on the oxidation of 0.2 μM UA is shown in The effect of the amount of BNNS-Pt NPs on the UA oxidation has been optimized. As shown in pa (μA) = 0.24221 c (nM) + 2.4454. The LOD can be calculated to be 0.5 pM (S/N=3).Under the optimum conditions, SWV was used for UA analysis. The reproducibility of the proposed electrochemical sensor was investigated by the detection of 0.2 μM UA with eight individual detections. A RSD of 4.1% was recorded, indicating that the BNNS-Pt NP dispersion exhibits good reproducibility compared with previous BNNS based works  and anions  method was also conducted for comparison.In this work, Ligustri lucidum fruit.Deposition of Pt NPs on a BNNS surface can be used for UA electro-oxidation. The BNNS-Pt NPs nanocomposite exhibited electrocatalytic activity toward UA oxidation. Loading of Pt NPs on BNNS surface can provide more electrocatalytic sites by exposing individual Pt NPs to the analyte without agglomeration. The proposed UA sensing platform showed a linear detected from 1 to 1,200 pM, with a low LOD of 0.5 pM. In addition, the sensing platform has been successfully used for determining UA content in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YF and JZ conducted the main experiments and wrote the paper. ZW did the material characterization and proofreading.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.


These questions are ill-posed. We submit that the way to tackle such questions is to adopt a biological approach, based on the evolutionary past of hominins. Hunter-gatherers in tundra or savannah environments have various, mutually distinct uses for color. Color differences aid in segmenting the visual field, whereas color qualia aid in recognizing objects. Classical psychophysics targets the former, but mostly ignores the latter, whereas experimental phenomenology, for instance in color naming, is relevant for recognition. Ecological factors, not anatomical/physiological ones, limit the validity of qualia as distinguishing signs. Spectral databases for varieties of daylight and object reflectance factors allow one to model this. The two questions are really one. A valid question that may replace both is how many distinguishing signs does color vision offer in the hominin Umwelt? The answer turns out to be about a thousand. The reason is that colors are formally not object properties but pragmatically are useful distinguishing signs.How many colors are there? Quoted numbers range from ten million to a dozen. Are colors object properties? Opinions range all the way from  It was upon a Sommers shynie day,When Titan faire his beames did display,In a fresh fountaine, farre from all mens vew,She bath’d her brest, the boyling heat t’allay;She bath’d with roses red, and violets blew,And all the sweetest flowres, that in the forrest grew.        Sir Edmund Spenser The Faerie Queene (1590)        Book Three, Canto vi, Stanza 6 .colors are object properties. The aim of this article is to explore the validity of this folk wisdom. The answer turns out to be implicate.In the aforementioned verse, the author silently assumes that Colorimetry was created by Remarkably, the formal structure is almost perfectly linear, indicating that it is due to elementary physical processes rather than complicated (and most likely nonlinear) physiological, or even psychological ones. Indeed, the theory is rooted in the physical process of the absorption of photons by three distinct pigments. No neural transformations or psychological processes are involved at that stage. So any properties decided at the earliest stage do not depend on processes at higher stages of the nervous system.1 involves a linear operator2  acting on the space of radiant power spectra , a fact known as color as an equivalence class , and later Helmholtz.8 In this article, we mainly use an extended construction, specifically targeted at object colors, which is essentially due to Perhaps the best known, basic colorimetric structure is the locus of colors defined by monochromatic beams. It is a convex conecie) xyz color matching functions as a numerical representation of the linear operator for the generic human observer  and (colorimetric) colors. It is closely similar to the system of generic red–green–blue (rgb) display colors and therefore more intuitive  nonsense. Spenser’s verse is mere artistic fancy. We formally prove this in Appendix C. One might not initially understand the theorem when formulated in terms of colorimetry proper, for there would be no mention of “white light” and “hues,” and there would be no names for qualia such as “red” or “blue.” There would just be illuminating beams A perhaps counterintuitive prediction of basic colorimetry is that it allows one to prove that white light, the rose and violet color change appears magical and implies that colors are not object properties. The formal proof is constructive, thus beyond doubt.An interesting interpretation of one formal theorem is that colors  are not object properties (in the sense of physics). It is indeed possible to design artificial roses, violets, and two radiant sources such that these (fake) roses are red, and these (fake) violets are blue as seen under one source (of fake daylight), whereas the same roses are blue, and the same violets are red under the other source (of another fake daylight). This pertains, even though a  daisy would look the same under either fake source, that is, “white,” as it looks under real daylight. Because it is thus visually obvious (the daisy is proof!) that both artificial sources provide blind spots  in the optical processing of spectral compositions.Notice that this is a purely colorimetric/physical fact. It simply exploits the human achromatic (lack of any particular hue) radiation. There is simply no way to handle this colorimetrically, as this does refer to phenomenology. This is very annoying. One tends to assume (silently referring to phenomenology) that the monochromatic beams have mutually distinct hues.12 But then, 13Other problems with basic colorimetry have to do with the nature of 14 Thus, there really has to be a colorless object color. Fortunately, in the context of object colors, one may call physics to the rescue.It is even more pressing for the object colors, because phenomenologically the most vivid object colors lie on a topological (full!) circle.white without reference to phenomenology. This necessarily involves object color. We are not aware of any such arguments that might help define achromatic beams. In the latter case, all one might do is to arbitrarily point out a certain beam as achromatic by decree.15Although not strictly colorimetry, there certainly exists a principled way to define object color also implies being seen as an object (or part of an object). This is by no means guaranteed. It requires an ecologically valid context.We skip a thorough discussion of phenomenology here. It should be understood that without any spectrally selective process. That is obviously a necessary condition for achromaticity and one that singularly applies to object colors.An obvious definition of a white object would require it to at least scatter all incident radiation The requirement is by no means sufficient. It would include perfect mirrors and diffraction gratings. A mirror has no color, and the diffraction grating has all colors. A mirror may take on any color, depending on what is reflected in it. A diffraction grating shows different colors as you view it from different directions.visual object. If this seems odd, then remember that a mirror is a physical object, but by no means a unique visual object. It lacks a color. A mirror may appear in any color, depending on what it incidentally happens to reflect.An additional requirement is that a white object should scatter the same beam to the eye, irrespective of where the eye is with respect to the object. This invariance might be taken as a definition of a Lambertian surface of unit spectral albedo. This definition does in no way refer to particular materials. It is very generic.Technically, this implies that the white object has to be Although our “white object” is a purely formal one, it is also necessary to have white reference objects in radiometric laboratory practice. In practice, white chalk or blotting paper come close. Pressed baryte .The definition of the white object is simply physics. It does in no way refer to particular materials. It is generic and unique. In the laboratory, one has to be satisfied with material implementations such as black velvet.16 Not perfect, but often good enough.17A black object is even simpler to define than the white object. It is a surface that does not scatter any radiation in any direction. Thus, an aperture that opens into infinite empty space  would be perfect. Again, this definition is simply Thus, the realm of object colors readily admits of a number of concepts—such as black and white—that greatly increase the use of colorimetric methods. It imposes fundamental constraints that the colorimetry of radiant beams lacks.appear white. But this will not work. In practice, one finds that white objects appear white under a wide range of colorimetrically distinct beams. People tend to use contextual information. This makes excellent biological sense. Consequently, colors as appearances do not uniquely correspond to colorimetric coordinates. Indeed, from an ontological perspective, colors as appearance and colors as colorimetric objects are mutually distinct entities.One might attempt to define an achromatic beam phenomenologically as a beam that makes a white object standard . Thus, perhaps the major importance of color is in locating key objects for bare survival , or have one Objects in real scenes may have a schizophrenic being, like a face seen en face may appear different from the same face seen en profil. The realization that Hesperus is Phosphorus  objecthood becomes void, as in the case of mirrors.18osphorus  is of a cie illuminant d65 average daylight  look white. This requires a generic context. When we refer to the spectrum of the source, we refer to the spectrum Consider a standard source. The source needs to have spectral radiant power over the full visual range. In our examples, we use cie d65 . The colArbitrary Lambertian sources cannot scatter more radiation than the white reference. Therefore, we consider radiant spectra hypercrate20 in the space of radiant spectra. It is convex and centrally symmetric. Because colors derive from a linear transformation (a projection) of the space of radiant beams, convexity and central symmetry are conserved  can be written as the color of rR + gG + bB, where rR + gG + bB will be referred to as the canonical representation of the color. The spectrum that gave rise to the color is metameric to the canonical representation.Denoting the parts r, g, b} is the unit cube rgb cube.” The vertex {0, 0, 0} represents black (abbreviated k). The vertex {1, 1, 1} represents white (abbreviated w).The domain of the coefficients {cardinal colors. The primary colors are {1, 0, 0} (r for red), {0, 1, 0} (g for green), and {0, 0, 1} (b for blue), whereas the secondary colors are {0, 1, 1} (c for cyan), {1, 0, 1} (m for magenta), and {1, 1, 0} (y for yellow). The cardinal colors occur in the natural periodic sequence ygcbmr.23 They are indeed “cardinal” because the only rgb cube points that lie on the boundary of the color solid. Proceeding from primary and secondary, we find that white is the unique tertiary cardinal color. Going in the reverse direction, we may add black. By construction, the pairs rc, gm, by, as well as kw are mutually supplementary (add to white). Standard colorimetry would reckon these “complementary with respect to the illuminant”; it has no natural notion of supplementarity.The remaining vertices may be divided into primary and secondary rgb cube lie on the surface of the color solid. The color solid (which is necessarily convex) appears as a slightly inflated unit cube  from 0 or 1. From a formal perspective, the rgb cube is the natural arena for the object colors .The construction of Again, the standard setup, that is the simplest possible setting, considers colored papers, laid out flat on a tabletop and illuminated by a uniform source, such as the northern sky, the classical illumination of artist’s studios .anchor the gamut (Let the object gamut contain a white object. It serves to he gamut . This isi ranging over the various objects). Then, the beam scattered to the eye by the 25 One also finds the parameters object color defined by the reflectance iF. The division implements “color constancy”  settings considered here, this is exceedingly rare (see later).When the spectrum of the illuminant is changed, the color solid itself changes, and the inscribed crate can no longer be the largest one. It will remain very close to that for relatively minor spectral variations, though. Colors might conceivably move in or out of the How descriptive this model is can only be judged on the basis of variations that remain in the ecologically valid range. Thus, the issue has to be investigated on the basis of statistical models of the generic environment .Umwelt  and Lebenswelt (or lifestyle)28 28 see , 2010.Colorimetry as such has nothing to say about the structure of color space, except that it is a linear space. Color space is just a projection of the space of spectra.psychophysics. For instance, one might measure hue discrimination , anthropocentrism implies dubious science.Exceptions that we ignore are such radiant sources as the bioluminescence of glowworms, fire light, and so forth. Glowworm light is a vital issue in the glowworm world, even if it is a mere curiosity in ours. Different animals, different realities. There are two aspects to this: the Umwelt and the lifestyle. The Umwelt contains all the ways the environment can work on the animal. For humans, this includes radiation in the visual band but excludes radio waves. It also includes all the ways the animal can work on the world. For humans, this includes moving stones but excludes generating strong electric fields without assisting technology.Animals may live in very different Umwelts. The lifestyle involves the use of Umwelt elements in the course of generic actions . For insThe ecologically valid realm of illuminants is evidently the range of daylight spectra. Hominins might have known fire light as a case of secondary importance. Yet it is the daylight—hunting–gathering time—that would have driven their evolution.Fortunately, there exist databases that cover daylights under parametric variation of:and a few more. Because it is awkward to mix databases, we use just a single database from the Kohonen laboratory . It shouThis database may well be slanted toward extreme cases. Anyway, the major influence of the illuminant is by way of spectral slope (color temperature) and  spectral curvature. The more or less random component due to environmental factors  hardly pThe primordial object for the terrestrial, diurnal animal is a volumetric region  bounded by an opaque, approximately Lambertian scattering surface. Think of a potato. Local surface elements of such objects scatter the same radiant beam to the eye from wherever the animal might be with respect to the object. The large majority of biologically important objects in nature are of this generic type. These include stones, earth, vegetation, animals, and processed things such as roots, wood, bones, meat, fat, and more. Even huge areas in the visual field such as the sky or large expanses of water fall in a different class. Their colors tend to be of little interest when you are hunting, or gathering, for your daily meal.has no color. The beam scattered to your eye depends critically on your position. In a hall of mirrors you are lost, albeit differently from being lost in the woods. In a hall of mirrors you may run into the walls, but in a forest, there is no reason to run into tree trunks. Tree trunks are bona fide visual objects, whereas mirrors are not.Common exceptions from our contemporary Umwelt tend to be man-made; think of polished metals. They came into existence after the evolution of hominin vision was already well on the way to its current state. A mirror It is difficult—indeed, probably impossible—to define an ecologically valid, statistically balanced, generic range of objects. It should certainly include:A study of spectral reflectance factors suggests that the spectral articulations found in these classes are statistically very similar.30,31 The vegetation database has 219 items: leaves, stems, flowers, and dry brown leaves. The mineral pigments database has 723 items of mainly classical painter’s pigments in powdered, dry form. After deleting samples with a significant specular component, 651 items are left. We let these represent the “earth colors” as different from the vegetation colors. Perhaps unfortunately, we are missing animal colors. There appears to be no suitable databases in the public domain. A spot check on spectral reflectances of bone, fat, meat, and skin suggests that it would hardly change the picture much, if at all. We feel that we cover the realm sufficiently.For a start, we select two databases: one on vegetation  and one Perhaps regrettably, we have no data on the frequency of occurrence or on the relative relevancy for the hunter-gatherer lifestyle. Our choice is likely to overrepresent singular outliers. But perhaps this is not such a great disadvantage. We are likely to obtain a somewhat pessimistic view of the ecological graininess.rgb cube more or less homogeneously  in which the green coordinate is less than rgb representation is so useful.32A very straightforward investigation simply puts all objects under all illuminants. Thus, we obtain 52 colors for each object. To streamline the presentation, we summarize these sets by covariance ellipsoids at the 95% level .We find that the automatic white balance works really well. This perhaps reminds one of results obtained by hyperspectral imaging . Althougcolor constancy, cannot be accounted for by the observer on the basis of available visual observations. They are due to hidden variables and thus naturally occur magically.The capricious variations of color appearances, noticeable as deviations from equivalent radius of an ellipsoid as the radius of the sphere with the same volume as that ellipsoid. We also define the maximum radius as the maximum semiaxis of the ellipsoid.We define the For the vegetation database, the range for the equivalent radius is rgb cube; thus, they are quite small. The mean ellipsoid volume is only rgb cube. This implies a Weber fraction per rgb coordinate of a few percent (take the cube root); thus, it exceeds the psychophysical discrimination level. It provides for about 20,000  distinct colors, much higher than numbers from experimental phenomenology (about a thousand).These numbers are in terms of the edge length of the For the mineral pigments database, the range for the equivalent radius is The confusion ellipsoids are evidently not isotropic. There is apparently an overall trend, although there is hardly a smooth field on the local scale. One reason for a trend is probably the fact that the daylight spectra differ mainly in one, at most two dimensions . The more or less abrupt variations are expected. They are doubtless due to the idiosyncrasies of the object spectra. These differ from the corresponding canonical spectra in manifold ways. One expects that taking the metamers of the object spectra into account would tend to smooth things out.Given a color, determine the ecologically valid set of  spectra that will yield this color under the standard illuminant;Determine the color of each of the metamers as seen under all ecologically valid illuminants;Summarize the resulting distribution, for instance, as a covariance ellipsoid;rgb cube.Repeat this for all colors in the Both conceptually and technically, metamerism poses a problem. It is quite clear what has to be done in principle though, namely:Problems are that the color is not likely to be in the database and that the database will not yield a set of representative metameric instances. For investigations of this type, the existing databases are hugely insufficient. The only way out is to summarize the databases in terms of generative statistical models .Of course, setting up such statistical models is a worthwhile scientific enterprise by itself. It has to be one of the fundaments required in studies of the evolution of the human visual system.To be able to proceed, we first need to consider some basic complications one immediately runs into in an attempt to implement this program. These mainly involve methods to deal with the nature of the domains. Such methods should not rely solely on formal methods. They need to be rooted in concepts from basic physics.physical entities.The computational machinery of formal colorimetry is linear algebra. This cheerfully operates with entities that have no possible physical existence. Such entities (such as the elements from the null-space of the color matching matrix) may well represent very useful virtual objects. However, eventually both the input and the output of computations need to represent Physics introduces a number of constraints on the objects that formal colorimetry works with. In a more positive vein, the physical constraints yield very important structures. The color solid itself is a prime example of that.Failure to reckon with physical constraints easily leads to erroneous results. A common instance involves nonphysical predictions being “cured” by clipping. Principal components analysis (PCA) is often made to work that way.ever obtain nonsensical results. In most cases, the solution is to use some variety of homomorphic filtering  domain, operates on it , and transforms back.Radiant power is necessarily nonnegative. An obvious way to deal with this is to consider the logarithm of radiant power . This is very natural from the perspective of physics. Most interactions involving radiant power are of a multiplicative nature. Another reason is that the fully noninformative Bayesian prior is hyperbolic, therefore uniform on the log scale , 1973. Tmetric. In using PCA, a default metric tends to be silently . Although the raw pixel histograms tend to be complicated, often bimodal, the histograms in the physical domain tend to look much more unimodal and symmetric . It is apparently the more appropriate domain for statistics. This is another instance of homomorphic filtering. In any case, one tries to map from the ecological to some appropriate physical domain.One may meet with a variety of physical processes. In any specific case, one identifies the most appropriate homomorphic representation. This calls for a broad familiarity with classical physics –1966.For the purpose of colorimetry in the context of the hominin Umwelt, the absolute levels of radiant power are largely irrelevant. It is mainly the spectral distributions that count. So the first thing to do is to somehow normalize the individual items, perhaps by shifting them in the physical domain.All instances are eventually due to sunlight, filtered by the atmosphere and affected by ambient scattered radiation. These are all multiplicative processes that become additive in the physical domain.Sunlight derives from the sun’s photosphere and is essentially thermal radiation of a Time of day and height above the horizon are expected to lead to changes of overall slope and perhaps curvature. These are most properly caught by the low-order PCs. Remaining variations are likely to be due to a variety of random causes. These are most appropriately modeled by some fractal signal. There is perhaps something to say for using Planck’s formula for thermal radiation augmented by a low-order polynomial model for the deterministic part. For many applications, that would avoid overfitting. In our application, the microarticulation had perhaps better be retained. They might accidentally correlate with microarticulations in spectral reflectance factors.33,34 We use the ensemble mean, a linear combination of the PCs with coefficients generated by a multinormal distribution (using the distribution of projections of the database instances on the PC-basis to define the covariance matrix), and a fractal fuzz. Finally, we transform to the ecological domain.The main decision is the level at which the deterministic (PCA) low order should give way to some random fractal. It appears to be the level after the PCs that essentially represent curvature or perhaps cubical structure. This yields a simple model that may serve to generate novel instances of “daylight spectra” .33,34 WeThe nature of the “fractal fuzz” is estimated from the slope of the power spectrum of the residual structure, after subtracting the PCA approximation.is important. Thus, we do not normalize in any sense. We just move to the physical domain, assuming radiative transport in turbid layers as the dominant process. There is no a priori reason to expect a bias in spectral slope or curvature. One expects the spectral articulation to be translation invariant in a statistical sense. The reason is that the visual range is very narrow and does not involve switchovers between distinct physical processes. Molecular mechanics dominates the infrared, atomic electronic transitions the ultraviolet, whereas the visual range largely involves various changes in chemical constitution induced by the impinging photons . The reason we do not use this huge hyperspectral database further is that this database seems very uneven and is likely to be troubled by systematic errors pertaining to large subsets of items.The behavior near the Because the flower spectra look rather diverse, a generic model might simply be a random fractal. We find the Fourier power spectrum of the spectral articulation  mutually independent physical causes. Each process will imprint a local structure on the spectrum at some random location. For the present purposes, the spectral width of the local structures is the relevant property. For a start, one considers just a single width and a random, uniformly distributed location. A simple, generic model of this physics is provided in Appendix B . This implies that natural spectra are not highly articulated. They vary only gradually with wavelength.36,37 One generates a fractal and adjusts its variance to that encountered in the database. One adds either the database mean, or just a constant slope. Converting from the physical to the ecological domain then yields the instance. As seen in rgb cube. This is only natural  solution is to limit the investigation to colors within the convex hull of the database colors. From a pragmatic perspective, this is not even such a bad idea. Colors far outside the convex hull are likely to be irrelevant anyway.For more formal investigations, a way out of this problem is to generate metamers as metameric variations of canonical spectra. Because there always exists a canonical instance, this is guaranteed to work for all colors. All one needs to do to generate metameric variations is to add random metameric blacks to canonical spectra. Of course, such metameric blacks should themselves be ecologically valid.38 Unfortunately, this tends to lead to unreasonably large amplitudes near the spectrum limits , the maximum amplitude of a perturbation is limited by that  and determine the covariance ellipsoid of the resulting set of colors that surround the fiducial one.Repeating this procedure for many colors, we obtain a sampling of the field of covariance ellipsoids over color space . This deThe mean ellipsoid volume is rgb coordinate of about 7% to 8%. This far exceeds the psychophysical discrimination thresholds but is comparable to estimates of color reproduction from experimental phenomenology  that consider only the segmentation function. They disregard the function of color as label, or as distinguishing mark. The jnds are likely due to physical (photon noise) and physiological factors. A number like 106 is at least three orders of magnitude larger than the grain size suggested from our investigation of the ecology.Psychophysical grain size data show that humans may resolve in the order of 107 colors . Such psvery much larger. We found that humans resolve about 103rgb colors if they are forced to use colors as distinguishing marks. Physical or physiological factors cannot be responsible for this grain size. These data address the factors of biological relevance . This renders the number intuitively acceptable.The precise distribution of the covariance ellipsoids found in our study  is not c3. This seems amply sufficient for most hunter-gatherer needs. Colors as distinguishing marks are really useful in the ecological setting, although they are formally proven useless in general.The grain induced by ecological variations limits the number of colors that might be used as distinguishing marks under all circumstances to about 10Are colors object properties? Our findings immediately relate to this question. It addresses an issue that has been widely debated in philosophy  anticipation. In biology, “object properties” can be more or less; they are not like the philosopher’s all or none. Nor deals biology with absolute truth. Evolution is driven by statistics, not  logical values.No distinguishing mark is expected to be decisive in isolation, except—perhaps—in cases of the ethological releasers. An example is the impact of the red of the male stickleback belly for the female stickleback . TypicalHow many distinguishing marks does one need anyway? If two berries have the same color, you may always consider the habitus of the branch from which they hang. Distinguishing marks are usually used in connection with one or two (independent!) others. Coincidences of relative rare marks soon amount to practical certainty; for small probabilities of mutually uncorrelated instances, multiply.hidden variables that are the spectral articulations not resolved by the color matching functions. In the absence of God’s Eye’s view , there is no way to reckon with that. The reason is the nonlinear (multiplicative) relation due to surface scattering. This magically promotes two mutually distinct sets of hidden variables into the observable domain.The metameric variations of “the” color of an object do not find a possible explanation in the agent’s Umwelt. They are due to limited articulation. The spectral correlation is appreciable over the visual range. Even more important, source and reflectance spectra are essentially never mutually correlated. This is due to their distinct physical origin. Thus, the major premisses that enable the proof in the Appendix C are rendered ineffective due to ecological factors. The magical effects of the hidden variables are (fortunately!) only minor.There evidently is something fishy about the general, “perfectly conclusive” proof. The reason is twofold. Both source and reflectance spectra are of works.In the final instance, this is why human color vision We have outlined a set of methods and a framework that enable one to study the influence of ecological factors on the utility of colors as distinguishing marks of significant objects in the hunter-gatherer tundra or savannah environment. That is the context in which the hominin visual system developed. The methods are fully general and can immediately be applied to any species, not just mammals.Such methods allow a perspective that has been sadly underrepresented in the literature of (human) color science. For instance, the fact that human observers are able to discriminate over a million colors is enthusiastically cited, but it gains a novel (in-)significance in view of data such as presented in 43 This is a topic that we unfortunately were not able to address.The applicability of these methods depends critically upon the availability of relevant databases. Unfortunately, there is not much to choose from, and novel data are not readily forthcoming. One wishes that hyperspectral imaging techniques will change that. This would also yield a possible handle on the ecological/ethological weights.If the desired data are not forthcoming soon (which appears likely), the only way to progress appears to be the development of generic models of source and reflectance spectra. This appears a definite possibility in view of such findings as shown in


Approximately one-tenth of the 10 million individuals living with human T-cell leukemia virus type-1 (HTLV-1) worldwide live in Japan. Most of these infected individuals live in the southwest region of Japan, including Okinawa prefecture; however, currently no prophylactic vaccine against HTLV-1 infection is available. For preventing the HTLV-1 spread, we previously generated a humanized monoclonal antibody (hu-LAT-27) that mediates both neutralization and antibody-dependent cellular cytotoxicity (ADCC). The neutralization epitope of LAT-27 is a linear amino acid sequence from residue 191 to 196 (Leu-Pro-His-Ser-Asn-Leu) of the HTLV-1 envelope gp46 protein. Here, we found that the LAT-27 epitope is well conserved among HTLV-1 clinical isolates prevalent in Okinawa. The hu-LAT-27 treatment inhibited syncytium formation by these clinical HTLV-1 isolates. Although an amino acid substitution at residue 192 in the LAT-27 epitope from proline to serine was found in a few HTLV-1 isolates, hu-LAT-27 could still react with a synthetic peptide carrying this amino acid substitution. These findings demonstrate the wide spectrum of hu-LAT-27 reactivity, suggesting that hu-LAT-27 may be a candidate drug for prophylactic passive immunization against HTLV-1 infection. The number of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals worldwide is estimated to be around 10 million ,3,4,5. HThe HTLV-1 envelope (env) spike consists of two glycoproteins, cell surface gp46 and transmembrane gp21, and is made up of a trimer of heterodimers . On targNevertheless, validating the efficacy of hu-LAT-27 requires extensive studies, including the determination of whether the LAT-27 epitope is well conserved in clinical HTLV-1 strains. Certain HTLV-1 stains isolated in the Caribbean, French Guiana, Gabon, and Brazil carry an amino acid substitution of proline to serine at residue 192 (P192S) in the LAT-27 epitope region ,24,25. ILAT-27 inhibits HTLV-1 transmission by binding to the neutralization epitope spanning the gp46 amino acid sequence from residue 191 to 196 . Among tThe present experimental conditions may have limited the possibility of detecting intra-host polymorphisms of HTLV-1 since the LAT-27 epitope region was analyzed using established cell lines. To address this issue, we further examined this region of the LAT-27 epitope using genomic DNA from fresh PBMCs from acute ATL patients , and found no intra-host polymorphism B. These The expression of the LAT-27 epitope in HTLV-1-infected cell lines was further examined using flow cytometry (FCM). HTLV-1-infected cell lines were treated with prostaglandin E2 (PGE2) to induce the maximum expression of HTLV-1 antigens, including gp46 and Tax [To confirm the neutralizing potential of hu-LAT-27 against these clinical HTLV-1 isolates, we performed a syncytium inhibition assay. PGE2-treated-HTLV-1-infected cell lines and an HTLV-1-uninfected target cell line, CEM, were co-cultured with or without hu-LAT-27 for 24 h. Syncytium formation was completely inhibited by the addition of hu-LAT-27 (+ T-cells during the 18 h of cultivation [We further investigated the conservation of the LAT-27 epitope among HTLV-1 produced by primary PBMCs and lymph node (LN) cells from patients with various subtypes of ATL or from asymptomatic carriers  using FCtivation . There wAn amino acid substitution from proline to serine at position 192 (P192S) in the LAT-27 epitope has been reported in Caribbean, French Guiana, Gabon, and Brazil isolates ,24,25. TIn this study, we demonstrated that the prototype of the gp46 amino acid sequence from position 191 to 196, namely Leu-Pro-His-Ser-Asn-Leu, in strain MT-2, which is recognized by LAT-27, is highly conserved in the current Okinawan clinical HTLV-1 isolates. Accordingly, all the clinical HTLV-1 strains tested were neutralized in vitro by hu-LAT-27, as shown by a syncytium formation assay. Furthermore, LAT-27 staining was detected in primary cultured PBMCs from patients with various subtypes of ATL and asymptomatic carriers. Notably, hu-LAT-27 could also bind to a synthetic gp46 peptide containing an amino acid substitution from proline to serine. Collectively, these results suggest that hu-LAT-27 may be effective in preventing the horizontal transmission of a broad range of HTLV-1 strains in vivo. The conservation of the LAT-27 neutralization epitope comprising six amino acid residues of gp46 has been reported for strains in other endemic areas, such as Romania, Zaire, Liberia, Melanesia, Australia, and Bellona . On the HTLV-1 shows remarkable homogeneity across subtypes, as its genetic diversity is known to be significantly lower than that of another RNA virus, human immunodeficiency virus type 1 (HIV-1). HIV-1 progressively expands and acquires genetic mutations in the env gene due to the low fidelity of its reverse transcriptase, which causes genetic errors during the reverse transcription of the viral genome . AlthougThe importance of the conservation of the env gene is demonstrated using its mutants, which revealed comprehensively inserted amino acids . The envIn conclusion, the present results show that the HTLV-1 neutralization epitope of gp46 recognized by hu-LAT-27 is widely conserved in Okinawan clinical HTLV-1 isolates. In addition to its key function in the neutralization of clinical isolates, hu-LAT-27 also appears to mediate the Fc receptor-dependent elimination of HTLV-1-infected cells in vitro through ADCC . Our resHTLV-1-infected cell lines were generated from PBMCs and LN cells obtained from HTLV-1-infected individuals living in Okinawa prefecture, Japan. These cells were obtained with the approval from the Internal Review Committee of the University of the Ryukyus (permit no. 478 and 319-5). Written informed consent was obtained from the participants before their enrollment in this study. Details on the clinical status of the blood donors, including the diagnosis and ATL subtypes classified according to Shimoyama’s criteria , are pro6 cells/mL for 18 h, followed by staining with LAT-27 and Lt-4. ATL and the carrier samples, which were comprised of over 0.01% Tax-expressing cells, were selected for analysis, as shown in Rat anti-gp46 (LAT-27) and mouse anti-Tax (Lt-4) mAbs were established and purified in our laboratory ,39. ThesGenomic DNA was extracted using the QIAamp DNA Mini Kit  according to the manufacturer’s protocol. Isolated DNA was subjected to PCR with the following set of primers: forward, 5′-tctagtcgacgctccaggat-3′, and reverse, 5′-gcaagtataattagtgctttgtagg-3′. DNA fragments were cloned into pGEM-T Easy Vector System I (Promega) and three clones were sequenced per genomic DNA specimen using the SP6 primer. The HTLV-1 gp46 gene of the MT-2 isolate was used as a reference sequence (GenBank: J02029.1).HTLV-1-infected cell lines established from Okinawa inhabitants were pretreated with PGE2 for 24 h. These cells were then co-cultured with the same number of CEM cells in 24-well microplates in the presence or absence of 50 μg/mL of hu-LAT-27. After 24 h of cultivation, syncytium formation was microscopically observed using an inverted microscope at a magnification of 100×, as reported previously .ELISA was performed as described previously . Two HTLt-test was performed for statistical analysis. Values of p < 0.01 were considered statistically significant.A paired


Plasmodesmata connect the cytoplasm of neighboring plant cells across cell walls. In response to various signals, they open and close to connect and isolate cells. We have found that the plasmodesmal plasma membrane hosts a unique immune-signaling cascade—different from that in the surrounding plasma membrane—which leads to plasmodesmata closure and cell isolation upon chitin perception. This response is mediated by a specific receptor complex, which in turn activates an NADPH oxidase via a specific regulatory module. This work characterizes how a cell can produce a localized and specific response in a discrete membrane domain, identifying that there is microdomain specificity in immune signaling to a single elicitor and that cell-to-cell connections are independently controlled. The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response. An array of plasma membrane (PM)-localized receptors projects into the extracellular environment to perceive a multitude of molecular signals. Receptor proteins (RPs) and receptor kinases (RKs) commonly act in complexes to activate signaling cascades upon binding their cognate ligands, mediating responses to a range of developmental and environmental signals. Plant cells exploit these receptors to perceive and signal in response to the presence of microbes, making these receptors critical components of the innate immune system. Thus, the mechanisms of receptor activation and signaling are central to plant cellular responses.Medicago LysM-CONTAINING RECEPTOR-LIKE KINASE 3 (LYK3) is recruited to a PM microdomain during rhizobia infection  to establish signaling hubs that execute response outputs –3. This 4nfection , and thenfection .Arabidopsis CRINKLY4 (ACR4) and CLAVATA1 (CLV1) RKs form plasmodesmata-specific complexes , the tubes that bridge neighboring cells to establish the interconnected cytoplasm. While electron micrographs identify that the plasmodesmal PM is continuous with the PM, the protein and lipid composition of the plasmodesmal PM differs from the rest of the PM –12, iden1315omplexes , and, reomplexes , 21. WhiIn the context of microbial perception and immune signaling, we have previously identified specific machinery required for plasmodesmal responses: the LYSM-CONTAINING GPI-ANCHORED PROTEIN 2 (LYM2) is located in the plasmodesmal PM and mediates chitin-triggered plasmodesmal closure , and theIt is not clear how an independent signaling cascade is executed in the plasmodesmal PM distinct from simultaneous PM signaling. Here, we exploited the specific involvement of LYM2 in chitin-triggered plasmodesmal responses to investigate the mechanisms by which neighboring membrane domains can signal independently. We found that in addition to LYM2, chitin responses in the plasmodesmal PM genetically require two additional LysM-RKs, LYK4 and LYK5. LYM2 can associate with both LYK4 and LYK5, but we detected only LYK4 in plasmodesmata, suggesting that chitin-triggered plasmodesmal signaling is mediated directly by a LYM2-LYK4 complex. The dependence of plasmodesmal responses on LYK5 appears to rest on its association with LYK4 in the PM prior to chitin perception. Chitin perception by LYM2 triggers activation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) via a specific phosphorylation signature linked to calcium-dependent protein kinases (CPKs). Supporting this, CPK6 and CPK11 are required to mediate chitin-triggered plasmodesmal responses. Ultimately, this signaling cascade induces callose deposition and plasmodesmata closure. Our findings characterize how the plasmodesmal PM specifically executes an immune-signaling cascade, illustrating how a single ligand can trigger independent responses in different membrane microdomains and demonstrating that plant cells can compartmentalize different outputs within the PM.Arabidopsis LysM-RK family consists of five members: CERK1/LYK1, LYK2, LYK3, LYK4, and LYK5. To narrow down plasmodesmata signaling candidates, we performed RT-PCR to identify members of the family expressed in mature Arabidopsis leaves where LYM2 functions. Only transcripts from CERK1, LYK3, LYK4, and LYK5 were detected in mature leaves . As we previously showed that CERK1 is not required for chitin-triggered plasmodesmata closure  plants . Thus, LYK4 and LYK5 are required for chitin-triggered plasmodesmata closure and are candidate partners for LYM2-mediated signaling.We previously identified LYM2 as a glycophosphatidylinositol (GPI)-anchored LysM-RP that resides in the plasmodesmal PM . Ligand 23 closure , we assak3, lyk4 , and lykmeasured , showed cerk1-2 mutants also shows an increase in plasmodesmal callose deposition in response to chitin, consistent with the chitin-triggered reduction in GFP movement through plasmodesmata previously observed in this mutant .Having identified that LYK4 and LYK5 are required for chitin-triggered plasmodesmata closure, we examined their subcellular localization to determine if they are present at plasmodesmata. We generated translational fusions of LYK4 and LYK5 to RFP and, as previously observed , 28, theNicotiana benthamiana tissue and purified plasmodesmata. As a positive control, we used the plasmodesmal protein PDLP5-HA  assays. We coexpressed Citrine-LYM2 with LYK4-HA, LYK5-HA, or LTI6b-HA in ractions B. While LYK5, but its absence from the plasmodesmata, we tested its role in the association between LYM2 and LYK4. For this, we transformed Arabidopsis lyk5-2 protoplasts with Citrine-LYM2 and LYK4-RFP  gels when extracted from lyk5-2 protoplasts and following chitin treatment the dominant LYK4-RFP band detected from lyk5-2 protoplasts is ∼30 kDa smaller than the dominant band in the Col-0 protoplasts  of PM-localized LYK4-GFP was significantly reduced in the presence of LYK5-RFP, indicating an increase in FRET as expected for interacting proteins . The change in FRET efficiency induced by chitin is ∼3.4%, suggesting that in this overexpression system, the pool of RKs that dissociate is small (and why it is undetectable in the Western blot). Chitin treatment decreased the LYK5-RFP–induced reduction in fluorescence lifetime of LYK4-GFP, indicating reduced FRET between these two proteins. To localize the occurrence of FRET within the PM, we marked plasmodesmata by coexpression of LYK4-GFP and LYK5-RFP with Citrine-LYM2. When we compared τAv of LYK4-GFP in regions of interest (ROIs) within the PM and at plasmodesmata, we observed that τAv in the PM was significantly reduced by the presence of LYK5-RFP but that in plasmodesmata, it was not . These data support our finding that LYK5 is not present in the plasmodesmal PM and further suggest that chitin weakens the interaction between LYK4 and LYK5 in the PM, possibly by complex dissociation or a change in complex conformation.To further explore the role of LYK5 in plasmodesmal signaling and its association with LYK4, we explored the possibility that LYK5 directly associates with LYK4 in the PM. Immunoprecipitation (IP) of LYK4-GFP from membrane fractions of t manner A. LTI6b-t manner B. The flChanges in protein association might result in a change in mobility within the PM. To test this, we made fluorescence recovery after photobleaching (FRAP) measurements to quantify the mobile fraction of the receptor population in the absence and presence of chitin. The mobile fraction of LYK4-RFP and LYK5-RFP both increased significantly 60 s postbleaching in the presence of chitin relative to mock conditions D. By conN. benthamiana leaves, we measured the plasmodesmal index (PD index) of fluorescence intensity , suggesting that LYM2 accumulation is a genuine element of LYM2 signaling.Recent studies have identified that some receptors accumulate at plasmodesmata in response to stress , 21. UsiN. benthamiana, we determined the effect of overexpressing LYK4 and LYK5 on LYM2 accumulation (Using transient expression in mulation B. A chitN. benthamiana tissue. We observed that anisotropy (r) of Citrine-LYM2 is lower in the PM relative to freely rotating cytosolic GFP, inferring that homo-FRET and LYM2 association occurs in the PM . In this assay, lym2-1 mutants were able to close their plasmodesmata in response to H2O2, demonstrating that any role for ROS signaling in chitin-triggered plasmodesmata closure occurs independently, or downstream, of LYM2. By measuring chitin-triggered ROS production in N. benthamiana leaf discs, we also determined that Citrine-LYM2, LYK4-RFP, or LYK5-RFP induces enhanced ROS production , indicating that each of these LysM proteins can trigger ROS signaling and is functional as a fusion protein.Reactive oxygen species (ROS) are produced during immunity and can induce plasmodesmata closure , 33. Thurbohd mutant is not able to close its plasmodesmata in response to chitin treatment . This was supported by quantitative analysis of plasmodesmal callose that showed that rbohd mutants did not deposit callose at plasmodesmata in response to chitin . Thus, ROS produced by RBOHD are a critical component of the chitin-triggered signaling cascade that induces plasmodesmata closure.The rapid production of ROS in response to chitin is associated with the NADPH oxidase RBOHD . BombardArabidopsis, RBOHD is regulated by phosphorylation by several protein kinases . By contrast, RBOHDS343A/S347A and RBOHDS133A cannot close their plasmodesmata in response to chitin. These data implicate Ser347 and Ser133 in chitin-triggered plasmodesmal closure.In  kinases –36. Rece kinases , 37. The kinases , 35. To rbohd mutant, Col-0, RBOHDS133A, and RBOHDS163A produced significantly more chitin-triggered ROS, while neither RBOHDS39A/S339A/S343A or RBOHDS343A/S347A showed a difference in ROS production relative to rbohd , while the plasmodesmata of the cpk5 mutant opened in response to chitin . By contrast, bik1 mutant plants showed a normal plasmodesmal response to chitin. Demonstrating specificity to plasmodesmal chitin responses, cpk6-1 and cpk11-2 mutants produced similar levels of chitin-triggered ROS to Col-0 plants   and in tOur investigation into the mechanisms of chitin signaling in the plasmodesmal PM identified that the plasmodesmata-resident GPI-anchored protein LYM2 accumulates at plasmodesmata in response to chitin. LYM2 also exhibits greater homo-FRET in the plasmodesmal PM than in the PM, indicating it oligomerizes or clusters there, possibly to form a signaling platform. It is well established that many forms of immune signaling occur via the formation of supramolecular complexes such as signalosomes and inflammasomes that involve the oligomerization of cytosolic NLR receptors and downstream signaling components. Membrane-anchored receptors also form multicomponent signaling complexes –43 and, We found that, in addition to LYM2, the RKs LYK4 and LYK5 are also critical for plasmodesmal PM chitin signaling. For PM chitin signaling, a CERK1-LYK4-LYK5 tripartite complex has been suggested  . HoweverWith respect to the role of LYK5, our data demonstrate that LYK5 can associate with both LYM2 and LYK4 in the PM B and 3A cpk5 mutant exhibits reduced cell-to-cell movement of GFP in control conditions , suggesting that it functions in plasmodesmal regulation beyond the chitin-response context. It is possible that plasmodesmal signaling is dependent only on CPK-mediated activation of RBOHD or alternatively that other, uncharacterized residues targeted by RLCKs might also function in plasmodesmal signaling.We identified that downstream of these receptors, chitin-triggered plasmodesmata closure is dependent on specific phosphorylated serines of RBOHD that are different from those required for the CERK1-mediated ROS burst B. The seSI Appendix, Figs. S2 and S7) suggests that plasmodesmata might be sensitive to changes in CPK and RBOHD function in mock conditions. Comparing genotypes, our data suggest the possibility that CPK5, CPK11, and phosphorylation of Ser133 and Ser163 of RBOHD negatively regulate plasmodesmal closure in mock conditions. It is possible that different CPKs and phosphorylation events act specifically as positive or negative regulators of NADPH oxidase activity or that different modes of NADPH oxidase activity convey different signals. However, independent of the mechanism, the data suggest that calcium and ROS signaling tune plasmodesmal responses across different conditions. This mechanism of plasmodesmal regulation, the specificity of the roles played by different CPKs, and the range of stimuli to which it is relevant require further investigation.Given the complexity of factors that influence plasmodesmal permeability, we have given greater weight to comparisons within genotypes to identify a capacity for response than to comparisons of the magnitude of cell-to-cell movement of GFP observed between genotypes. However, examination of the raw data  is wild type. The mutant lines used in this study are rbohd . All annotated images were sanity-checked prior to inclusion in Dataset S1.Plasmodesmal callose staining and quantification were performed as described . Three zN. benthamiana transiently expressing the constructs of interest, or lym2/pLYM2::Citrine-LYM2 Arabidopsis plants, were infiltrated with chitin (500 µg/mL) or water (mock conditions) and stained with 0.1% aniline blue. The abaxial side of the leaf was imaged using a 63× water-immersion objective lens  with a confocal microscope. PD index was determined by measuring the intensity values of Citrine-LYM2 in ROIs that represent plasmodesmata and neighboring PM regions with ImageJ.Leaves of N. benthamiana transiently expressing Citrine-LYM2 and cytosolic GFP were imaged with a Leica SP8 confocal microscope. Citrine was excited with a pulsed white light laser (488 nm), and emitted light was separated into parallel and perpendicular polarizations and detected by external single photon avalanche diodes with 500- to 550-nm filters. A series of 20 frames was merged and analyzed using PicoQuant SymPhoTime 64. Anisoptropy (r) was calculated byLeaves of G = 0.481, L1 = 0.013, and L2 = 0.037. ROIs were defined that correlated to plasmodesmata and the PM for Citrine-LYM2 images or the cytosol for GFP images.where N. benthamiana transiently expressing the constructs of interest were used 30 min after infiltration with chitin (500 µg/mL) or water (mock conditions) for FRET-FLIM. The abaxial side of the leaf samples was imaged using a 63×/1.2 water-immersion objective lens . FLIM experiments were performed using a Leica TCS SP8X confocal microscope equipped with time-correlated single-photon counting electronics (PicoHarp 300), photon-sensitive detectors (HyD SMD detector), and a pulsed white light laser (470 to 670 nm). GFP was excited at 488 nm (40 MHz) and collected between 509 to 530 nm. Data analysis is described in full in SI Appendix.Leaves of SI Appendix.FRAP was performed using a Leica TCS SP8X CLSM with a 63×/1.20 water-immersion objective . ROIs were defined for plasmodesmata-localized Citrine-LYM2 and for PM-localized Citrine-LYM2, LYK4-RFP, and LYK5-RFP. Fluorescence recovery was measured in ROIs 60 s postbleach. Data analysis is described in full in Arabidopsis protoplasts and N. bethamiana leaves were used for immunprecipitation experiments. Proteins were extracted in IP buffer containing 50 mM tris(hydroxymethyl)aminomethane∙HCl (pH 7.5), 150 mM NaCl, 5 mM dithiothreitol, protease inhibitor mixture (Sigma) 1:100, phosphatase inhibitor (Sigma) 1:200, 1 mM phenylmethanesulfonyl fluoride, 0.5% IPEGAL CA-630 (Sigma), 1 mM ethylenediaminetetraacetic acid, 1 mM Na2MoO4 × 2H2O, 1 mM NaF, and 1.5 mM activated Na3VO4. For co-IP, GFP-Trap agarose or magnetic beads (ChromoTek) were incubated with the protein samples for 2 h at 4 °C with gentle agitation. Beads were washed at least three times with IP buffer and proteins released by heating to 95 °C in Laemmli buffer (2×).Arabidopsis suspension cultures cells , and luminescence was emitted in the first 25 min after elicitation was integrated, corrected for background luminescence, and used for subsequent analysis.P value was less than 0.05; n values are listed in SI Appendix, Table S3. Details for the design and statistical analysis of each experiment are detailed in SI Appendix, Extended Methods.Statistical analyses were performed using Genstat Version 18 or R Version 3.5.1. Unless otherwise indicated, statistical significance was concluded when the Dataset S1. R scripts are available in GitHub (All data measurements used in this paper are available in n GitHub .Supplementary FileSupplementary File