Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.—Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues,  Endogenous circadian rhythms enable organisms to anticipate and adapt to the predictable environmental changes that occur as a result of the 24-h light/dark (LD) cycle. As well as dramatic changes in activity and rest, rhythms exist in many physiologic parameters such as body temperature and hormone levels. The master circadian pacemaker is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus , and it The functions of CRYs in the retina have been the subject of considerable interest. Their role as photoreceptive molecules in plants and insects has led to speculation that CRYs may act as the retinal photoreceptors that detect light and transmit photic information to the SCN to entrain rhythms in physiology and behavior . The nat5Per1 . Effects on non–image-forming responses to light in double-knockout mice were taken as evidence that CRYs functioned as photopigments  Act 1986  and the University of Oxford Policy on the Use of Animals in Scientific Research. All procedures were performed in a designated establishment.Wild-type (WT) C57BL/6J,  Cry2−/− , and PER Cry2−/−  mice werRetinal sections were prepared as previously described . Tissue https://imagej.nih.gov/ij/). For quantitative comparisons, all images were collected and processed under identical conditions.Images were acquired as previously described . An LSM Whole-retina explants were prepared from adult mice of both sexes (>30 d old) and mounted on Millicell culture inserts (EMD Millipore). Retinas were maintained in culture for 24 h before recording bioluminescence rhythms as previously described . Whole-rn = 5; 3 male and 2 female), −/−Cry1  and −/−Cry2 . Contrast sensitivity, WT , −/−Cry1 , −/−Cry2 . Pupillometry, WT , −/−Cry1  and −/−Cry2 . Unless otherwise stated, each mouse was tested at all 4 testing times  in a random order. Finally, to reduce light adaptation effects on dark-adapted mice, testing periods for individual mice were restricted to less than 20 min from first exposure to light.Circadian physiology testing was performed after 1 d in constant darkness at circadian time (CT) 6 ± 1 h (subjective midday) and CT18 ± 1 h (subjective midnight). Furthermore, diurnal physiology recordings were made at midday [zeitgeber time (ZT) 6 ± 1 h] and midnight (ZT18 ± 1 h) hours in the LD cycle. Sample size and sex were as follows: electroretinography, WT  for 25 min in total. In a single case (−/−Cry1 mouse electroretinogram (ERG) recorded at CT18 after 17.5 min light adaptation), a single data point was unobtainable due to technical problems. The b-wave amplitudes were quantified with specialized software . At the end of the procedure, general anesthesia was reversed by intraperitoneal injection of atipamezole hydrochloride .Before electroretinography, general anesthesia was induced in mice by a single intraperitoneal injection of medetomidine hydrochloride  and ketamine , and pupils were fully dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride eye drops . Electroretinography recordings were performed as previously described  . PhotopiOptokinetic head tracking (an optomotor response test) was used to measure contrast sensitivity and visual acuity using the OptoMotry system  as previously described . The tes2/s (173 μW/cm2/s). Irradiance measurements were made using a radiometrically calibrated spectrophotometer . Light stimuli (10 s) were transmitted to the eye via a liquid light guide as an irradiant light stimulus using a 50.8 mm integrating sphere  and was controlled by a shutter positioned in the light path . Images of consensual pupil responses were collected with a Prosilica NIR-sensitive CCD video camera (BRSL) at a rate of 10 frames/s under infrared LED illumination . During pupil measurements, unanesthetized animals were temporarily restrained using normal husbandry techniques for the duration of the recording . Each animal was handled and pupil light responses recorded at least twice several days before the test in order to minimize any artifacts due to handling and procedure-related stress. All images were analyzed by ImageJ software.Pupil light responses were measured as previously described , 30. Forsem. Statistical analysis was performed by paired 2-way ANOVA with post hoc testing or paired/unpaired 2-tailed Student’s t test by GraphPad Prism 6 .Data are presented ± −/−Cry2 mice, suggesting a lack of specificity  as previously described . Labelincificity A, B. As cificity , 31 werecificity C, D. Theglutinin . Anti-he−/−Cry1 retinal tissue was used as a control and showed no specific CRY1 signal  exhibited a retinal circadian rhythm with a period of 23.9 ± 0.14 h, whereas −/−Cry1 mice (n = 11) had a retinal rhythm period of 21.9 ± 0.50 h and −/−Cry2 mice (n = 5) a period of 25.9 ± 0.11 h (n = 7) demonstrated an SCN circadian rhythm with a period of 24.0 ± 0.35 h, whereas −/−Cry1 mice (n = 7) had a retinal rhythm period of 22.1 ± 0.28 h and −/−Cry2 mice (n = 5) a period of 25.9 ± 0.30 h  = 8.77, P = 0.0014), and Tukey’s post hoc multiple comparisons tests revealed a significant difference in sem between WT and −/−Cry1, as well as between −/−Cry1 and −/−Cry2, but not between WT or −/−Cry2.When comparing the peak-to-peak period measurements of PER2::LUC bioluminescence rhythms in individual retina explants, we found that ant data G and groant data H show th retinas I. The meGiven the observed differences in retinal expression patterns and effects on retinal clock amplitude and stability, we proceeded to investigate whether CRY1 and -2 may have differential effects on the regulation of rhythmic retinal physiology. The photopic (light adapted) ERG b-wave amplitude has been reported as a reliable output of the retinal clock –37. Thisn = 5) showed a significant main effect of light adaptation time  = 39.0, P < 0.0001), which peaked at around 10 min  = 28.8, P < 0.0001), and a significant interaction between CT × light adaptation time  = 5.01, P < 0.0001). Post hoc Bonferroni tests showed that WT mice have significantly higher b-wave amplitudes during the subjective day (CT6) across the range of 5 to 12.5 min of dark adaptation. In −/−Cry1 mice (n = 7), significant differences of light adaptation time were still apparent  = 22.5, P < 0.0001), peaking at around 10 min, as in WT animals  = 3.1, P = 0.083) and no significant interaction between CT × light adaptation time  = 1.13, P = 0.358). Post hoc Bonferroni tests showed no differences between CT at any duration of light adaptation. −/−Cry2 mice (n = 7) showed a significant effect of light adaptation time  = 19.8, P < 0.0001), again peaking around 10 min  = 9.04, P = 0.0038) but no significant interaction between CT × light adaptation time  = 0.167, P = 0.997). Post hoc Bonferroni tests showed no differences between CTs at any duration of light adaptation. As such, while a significant effect of CT was apparent, this circadian rhythm appears to be attenuated compared to WT animals.Photopic light-adapted electroretinography was performed under constant darkness at CT6 and CT18 as in previous studies –37. ERG  animals B. Howeved 10 min C. HoweveA 10 min light adaptation protocol is most commonly used to study retinal rhythms, as this provides the maximal photopic b-wave amplitude , 37. WheNpas2, and adenylate cyclase 1. Because NPAS2 is only localized to the retinal ganglion cell layer, it appears that loss of the inner retinal clock alone is capable of abolishing rhythms in contrast sensitivity  = 0.630, P = 0.432) or genotype  = 1.27, P = 0.292), and no interaction between CT × genotype  = 0.120, P = 0.887). We then tested the contrast sensitivity of mice at CT6 and CT18 (n = 7), there was a significant effect of spatial frequency  = 39.0, P < 0.0001, F = 150, P < 0.0001), with higher contrast sensitivity during the subjective day (CT6). A significant interaction occurred between CT × spatial frequency  = 5.44, P = 0.0004). Post hoc Bonferroni tests showed that WT mice have significantly higher contrast sensitivities at CT6 at spatial frequencies between 0.064 and 0.192 cycles/degree. In −/−Cry1 mice (n = 10), the main effect of spatial frequency was still found to be significant  = 14.1, P < 0.0001, F = 27.2, P < 0.0001) compared to WT animals, contrast sensitivity in −/−Cry1 mice was higher during the subjective night (CT18). There was also a significant interaction between CT × spatial frequency  = 5.00, P = 0.0014). Post hoc Bonferroni tests show that −/−Cry1 mice only show a significant difference in contrast sensitivity at a spatial frequency of 0.272, but not at any other spatial frequencies tested. As such, the main effect of CT appears to be due to the significant difference at 0.272 cycles/degree (high acuity).Confirming previous data, we found that visual acuity did not show any diurnal or circadian variation between WT mice and mice lacking CRY1 or -2  again showed a significant effect of spatial frequency  = 26.9, P < 0.0001), peaking at 0.064 cycles/degree  = 91.2, P < 0.0001), with higher contrast sensitivity during the subjective day (CT6), comparable with WT animals. There was a significant interaction between CT × spatial frequency  = 4.73, P = 0.002). Post hoc Bonferroni tests showed that −/−Cry2 mice have significantly higher contrast sensitivities at spatial frequencies between 0.031 and 0.103 cycles/degree, comparable with WT animals.s/degree E. There When contrast sensitivity is compared at the peak of visual acuity, 0.064 cycles/degree as reported previously , signifin = 5), −/−Cry1 (n = 7), and −/−Cry2 (n = 7) mice . While no significant circadian difference was detected in −/−Cry1 mice , −/−Cry2 mice showed significant circadian differences, comparable to WT animals .The pupillary light response (PLR) exhibits both circadian and diurnal rhythms in response magnitude . We ther−/−Cry1 mice showed a PLR rhythm under ZT conditions, which is likely due to light masking, similar to −/−;Cry2−/−Cry1 mice, which show behavioral activity masking under LD conditions  is sufficient to induce arrhythmicity, while SCN rhythms are maintained in the absence of Per1 or Cry1, albeit with a change in period  show comparable responses to those observed in the subjective day (CT6). While loss of retinal rhythms may be expected to result in an averaging of daytime and nighttime responses, −/−Cry1 mice appear to display daytime responses at all phases. Similar findings have been reported in −/−;Cry2−/−Cry1 mice  show enhanced sensitivity to light in ERG and PLR responses, consistent with the role of Rev-erbα in providing negative feedback on BMAL1 and E-box activation. However, these data are additionally complicated by a role of Rev-erbα in regulating melanopsin expression  at 0.272 cycles/degree. Not only is this response the reverse of what is seen in WT and −/−Cry2 animals but it also occurs consistently under both entrained (ZT) and constant (CT) conditions, and only at the highest spatial frequencies . It is unclear whether the different retinal phenotypes observed in clock gene knockouts may relate to their role in the regulation of tissue-specific transcriptional profiles. Because CLOCK and BMAL1 are transcription factors that drive the tissue-specific expression of numerous other target genes, loss of these critical factors may produce effects independent of their role in the circadian clock (Our data also demonstrate that CRY1-deficient mice provide a valuable model to study the role of the retinal clock because, unlike an clock –53. As san clock , this ma−/−Cry1 mice show attenuated or abolished retinal rhythms, whereas −/−Cry2 animals only show deficits in ERG responses. These retinal functions can be correlated with the expression pattern of CRY1 and -2 in the mouse retina, with CRY1 expression detected in multiple cell types, whereas CRY2 expression is more limited. These findings suggest that CRY1 and -2 play distinct and separate roles in the regulation of retinal circadian physiology.In summary, our data show that circadian rhythms in retinal physiology depend primarily on CRY1, while CRY2 appears largely redundant. Using multiple assays of retinal circadian rhythms, we show that http://www.fasebj.org to obtain this information.This article includes supplemental data. Please visit Click here for additional data file.


Results indicated that irrigation triggered N2O pulsing effect in all SDI treatments, yet N2O values reduced with the decrease of surface soil wetting proportions of SDI irrigated soils, and the occurrence times were lagged. The peak N2O fluxes and the corresponding soil water filled pore space (WFPS), as well as the coefficients of determination (R2) of the exponential function between N2O fluxes and soil WFPS, decreased with the reduction of surface soil wetting proportions with SDI treatment, and from the central sub-region to the periphery sub-region. The pulse period contributed most to the reduction of N2O emissions in SDI compared to SI treatments and should be a key period for N2O emission mitigation. Over the whole experimental period, the area-weighted average cumulative N2O fluxes from SDI treatments were 82.3–157.3 mg N2O m−2 lower than those from SI treatment, with periphery sub-regions of R3 and R4  contributing to more than 75.8% of the total N2O emission mitigation. These results suggest that reducing surface soil wetting proportions or the increments of topsoil WFPS for SDI irrigated soils is a promising strategy for N2O emission reduction.To reveal the impact of soil moisture distributions on nitrous oxide (N It is aar scale . Accordimissions ,6.2O emissions from soils [2O within the soil. Generally, denitrification rates increase rapidly when water filled pore space (WFPS) exceeds 60%, due to the decrease of oxygen supply, and predominates over nitrification in the production of N2O [2O emissions when WFPS falls in the range of 30–70% [Water management has been recognized as one of the key factors that triggers Nom soils ,8,9. Soin of N2O . Yet, nif 30–70% . 2O. Under surface irrigation (SI), where water is applied evenly to the soil surface, soil moisture is expected to be distributed homogeneously. However, for partial wetting irrigation, such as subsurface drip irrigation (SDI) or drip irrigation (DI), water is applied by a point source emitter at a low rate [4+ and NO3−, and soil microbial community composition), and thus in the production and emission of N2O [2O emissions [2O emissions from different sub-regions around the emitter were investigated under a specific wetting pattern, and it was found for the first time that N2O fluxes decreased gradually from the central sub-region to the periphery sub-region around the emitter [2O emissions. However, N2O emissions from SDI irrigated soils under different surface soil wetting proportions are rarely mentioned, and N2O emission responses to soil moisture among different SDI wetting patterns are still unclear. Furthermore, soil WFPS is affected, both temporally and spatially, by the amount of irrigation water and its corresponding irrigation method, which inevitably affect the production and emission of Nlow rate , which glow rate ,14. The n of N2O ,16. It hmissions ,17,18. R emitter . For SDI emitter ,21, whic2O emissions from SDI irrigated soils under different surface soil wetting patterns, and to reveal the differences in N2O emission responses to soil moisture among SDI wetting patterns. Thus, pot experiments were conducted on a silty clay soil with three surface soil wetting proportions produced by SDI. The objectives were to quantify NSolanum lycopersicum L.) in the Vegetables Scientific Institute of Nanjing, China . Soil organic matter, total nitrogen, and total phosphorus were 25.2, 1.4 and 0.04 g kg−1, respectively. Soil ammonium nitrogen and nitrate nitrogen were 8.8 and 34.2 mg kg−1, respectively and the soil pHH2O was 6.2. Soil samples were air-dried, ground, sieved, homogenized, and finally packed into soil columns to the bulk densities of 1.25 and 1.33 g cm−3 for 0–10 cm and 10–50 cm layers, respectively, according to the local soil bulk densities. To avoid the influence of soil disturbance on soil N2O emissions, soil columns were left undisturbed for more than 60 days. During the experimental period, soil columns were placed outdoors in a completely randomized pattern and covered by an awning on rainy days only, to avoid the influence of rainfall.Experiments were conducted on a silty clay soil from August to December 2014 at the State Key Laboratory of Hydrology-Water Resources and Hydraulic Engineering in Nanjing, China . Air temperature during the experimental period ranged from 15.8 to 31.3 °C, and the precipitation was summed as 98 mm. Soil samples were collected in 2012 from a greenhouse after the tomato harvesting  were realized by placing SDI emitters at different depths  and supplying different amounts of water  [Soil columns were irrigated twice during the experimental period, at 10:00 h on 24 August and 9 October 2014. Soils in the SI, SDI–1, SDI–2, and SDI–3 boxes were irrigated with 7500, 3620, 2895 and 2500 mL of water, respectively, for the first irrigation, and with 7000, 3260, 2610 and 2200 mL of water, respectively, for the second irrigation. To map soil moisture distribution, soil samples were taken daily during the first 5 d and then at intervals of 3–5 d after each irrigation. For SI treatment, soil samples (approximately 3–5 g each) from depths of 0 to 24 cm in 4 cm increments were collected through holes (Inner diameter (I. D.) = 20 mm) located in one side of each soil box using a stainless steel sampler (I. D. = 18 mm). For SDI treatment, soil samples were collected in horizontal and vertical directions through holes (I. D. = 10 mm) located in one side of each soil box using a stainless steel sampler (Outer diameter (O. D.) = 8 mm), at 5 cm intervals from 0 to 55 cm layers vertically and 2.5 to 37.5 cm horizontally. Soil moisture content was determined gravimetrically by drying at 105 °C for 24 h, and WFPS was calculated as the percentage of soil volumetric moisture content  to soil porosity, with total porosity calculated from the soil bulk density and particle density  from each SDI soil column were collected using a static chamber  with an electron capture detector (ECD). Fluxes of N2O were calculated according to the linear increments of N2O concentration and the temperatures within the chambers [2O emissions from each sub-region were calculated by integrating N2O fluxes along the sampling period. Area-weighted average N2O emissions from each SDI treatment were calculated based on the cumulative emissions from sub-regions R1–R4 with percentages (surface area proportion of each sub-region to the sum) of 7%, 19%, 31% and 43%, respectively.Nchambers . Cumulatt-tests were performed to detect differences in N2O fluxes among SDI irrigated soils with different surface soil wetting proportions. Pearson’s correlation analyses were performed to investigate the relationships between soil moisture and N2O fluxes.Statistical analyses were performed using SPSS19.0 . Paired Soil moisture at 0–12 cm layers varied markedly in both the SI and SDI treatments , due to For SDI treatments, surface soil wetting proportions after the initial irrigation were 39.7% (SDI–1), 10.9% (SDI–2) and 1.7% (SDI–3), respectively. Soil moisture among the SDI wetting patterns showed a similar pattern, and soil wetting bodies were ellipsoid in shape with emitters at the center . Soil moFor treatment SDI–1, the soil WFPS was above 60% in sub-regions near the emitter during the first 48–72 h after water was applied, whereas at the wetting front (the interface between wet and dry areas) it was less than 40%. At 24 h after the first irrigation, the average WFPS in 0–20 cm of soil reduced horizontally from 82.3% at 2.5 cm from the drip emitter, to 25.4% at 37.5 cm from the drip emitter. Then, soil moisture in different sub-regions decreased with elapsed time, and reduced to 20.8%–63.0%, 11.5%–46.6% and 9.2%–39.8% at 240 h, 456 h and 816 h after the first irrigation, respectively. Soil moisture among sub-regions after the second irrigation showed a similar pattern to that after the first irrigation, although the area-weighted average (0–20 cm) WFPS was 0.2%–3.5% higher. Compared to SDI–1, soil moisture for SDI–2 and SDI–3 treatments showed a similar pattern, although the area-weighted average WFPS was reduced by 3.4–16.7% and 4.0%–15.4% after the first irrigation, and by 4.7%–21.3% and 4.2%–18.5% after the second irrigation, respectively .2O fluxes from SI treatment were maintained at a high level at the beginning  of the experimental period; following this, N2O fluxes reduced with the decrease of soil average (0–12 cm) WFPS  WFPS were within the ranges of 75.6%–85.1% and 74.5%–84.3%. Irrigation events resulted in pulse N2O emissions from SI irrigated soil. In addition, the peak N2O flux after the second irrigation was seven times greater than that at 816 h after the first irrigation (or prior to the second irrigation). Ncm) WFPS . The max2O fluxes from SDI treatments differed slightly among sub-regions and among surface soil wetting patterns  WFPS of 76.1% and 79.5% , with fluxes in sub-region R4 having the lowest. Also, soil WFPS in 0–20 cm layers corresponding to the peak N2O fluxes decreased from sub-region R1 to sub-region R4. Npatterns  and werepatterns . For SDInd 79.5% A. Then, 2O fluxes from SDI–2 and SDI–3 treatments varied in patterns similar to those from the SDI–1 treatment, although the peak times lagged by 0–24 h and 24–72 h, and peak values decreased by 17.4%–49.9% and 71.5%–77.0% (p < 0.05), respectively (2O emissions and the maximum soil WFPS were longer in SDI–2 (24–72 h) and SDI–3 (24–120 h) treatments than in SDI–1 (0–72 h) treatment.Nectively B,C. Mean2O emissions from SDI irrigated soils were positively correlated with surface soil wetting proportions, and these fluxes were reduced from SDI–1 to SDI–3 treatments. Within a specific surface soil wetting pattern of SDI irrigated soil, N2O fluxes decreased from the central sub-region to the periphery sub-region and the fluxes were significantly lower than those from SI irrigated soil, reducing on average by 33.5%–54.5%, 54.4%–64.7% and 77.3%–86.7% for SDI–1, SDI–2 and SDI–3 treatments, respectively. Among sub-regions, the differences in N2O fluxes were more marked during the pulse period than during the post-pulse period. In particular, soil average N2O fluxes from sub-region R4 were significantly lower than those from sub-region R1 during pulse periods  after the first irrigation (p < 0.05), yet no significant differences in N2O emissions were observed among sub-regions during the post-pulse period after the second irrigation. Generally, N2O emissions in all sub-regions from SDI–1, SDI–2 and SDI–3 treatments were within the ranges of 92.9–132.6, 70.0–89.6 and 25.5–43.7 mg N2O m−2, respectively, during two drying–wetting cycles, which were 29.4%–50.6%, 52.3%–62.8% and 76.9%–86.4% lower, respectively, than those from the SI treatment , with differences significant at the p = 0.05 level. Moreover, cumulative N2O fluxes in sub-region R1 were significantly higher than those in sub-region R4 of all SDI treatments (p < 0.05), but no significant differences were observed between sub-regions R2 and R3 (Cumulative Nreatment . The soi2 and R3 .2O emissions by 40.2%–63.0% (SDI–1), 58.6%–67.7% (SDI–2) and 77.6%–85.4% (SDI–3) in the pulse period, and by 103.1%–221.8%, 19.4%–38.2% and 3.0%–7.2% in the post-pulse period, respectively , 64.4% (SDI–2) and 85.4% (SDI–3) in the pulse period, and 43.8%, 59.8% and 83.7% during the two experimental periods, respectively. These results suggested that the pulse period contributed most to the reduction of N2O emissions between the SDI and SI treatments and should be a key period for N2O emission mitigation.Compared to SI, SDI decreased cumulative Nectively . Among S2O emissions  from all sub-regions were within ranges of 1.7–7.5, 1.1–5.6 and 0.5–2.1 mg for SDI–1, SDI–2 and SDI–3 treatments, respectively, and reduced from the central sub-region to the periphery sub-region for each specific SDI wetting pattern  of these exponential functions were within the ranges of 0.334–0.465, 0.708–0.864 and 0.671–0.723 for SDI–1, SDI–2 and SDI–3 treatments, with values in the sub-region R2 from SDI–2 treatment slightly higher than those from SDI–1 and SDI–3 treatments. For each specific SDI wetting pattern, the R2 decreased from central sub-region to periphery sub-region.Correlation analysis indicated that Noil WFPS . The coe2O emissions occurred immediately following heavy rainfall or irrigation, ranged from a few to 18,000 mg N2O m−2 h−1 [2O emissions [2O emissions from SDI irrigated soils with different surface soil wetting proportions. The magnitudes and the contributions  of peak N2O emissions found were consistent with the previous research. This was possibly because microbiological activity was very weak, and many bacteria and fungi died when the soil was dry which resulted in increasing concentrations of labile N and C. The addition of water  stimulated nitrification and de-nitrification processes and therefore increased N2O emissions [2O caused by irrigation events.Previous research showed that pulse N m−2 h−1 ,23 and cmissions . The premissions ,25,26. T2O emissions from soils [2O fluxes [2O emissions were generally observed when the WFPS fell in the range of 45%–90% [2O fluxes were mainly within the ranges of 22%–79% (SDI–1), 17%–59% (SDI–2) and 13%–44% (SDI–3)  , which wperties, ,32,33. 2O emissions were smaller and the occurrence times were later in sub-regions R3 and R4. This phenomenon may be attributed to the following reasons: Firstly, soil moisture (10%–80% WFPS) under partial wetting irrigation (where insufficient water was supplied) was generally maintained at an appropriate range for N2O emissions, and N2O fluxes generally increased with the increase of moisture content within the moisture range [2O emissions reduced sequentially; secondly, the soil with a high water content moved downward from sub-region R1 to R4  of the exponential functions, as well as the average slopes, were in the sequence of SDI–2 > SDI–3 > SDI–1 and decreased gradually from the central sub-region to the periphery sub-region. These results suggest that the degree of impact of soil moisture on N2O emissions might be slightly different either among SDI wetting patterns, or among sub-regions.Additionally, some research addressing the relationships between Noil WFPS ,30,31. Iterature ,29,30,312O fluxes in different sub-regions from all SDI irrigated soils were found to increase exponentially with the increase of the maximum soil WFPS (N2O peak flux = 24.8 e3.414 WFPS(Max), R2 = 0.663, p < 0.05, 2O fluxes are determined by the corresponding maximum WFPS values, even if the corresponding soil moisture level is lower than the previously reported optimal soil moisture level (45%–90% WFPS) for peak N2O emissions. This indicated that reducing the increment amplitude of soil WFPS in the topsoil of each sub-region was helpful for decreasing pulse N2O emissions. In addition, the decreased magnitude of peak emissions would be the final determinant of the total reduction potential of N2O emissions, as the reduction of N2O emissions in the pulse period contributed a larger proportion of the total N2O emission reduction  are applied widely to cope with water scarcity. For example, Sánchez-Martín et al. [2O emissions from horticultural soil and proposed that the drip irrigation method can be used to save water and mitigate N2O emissions. Kallenbach et al. [2O flux under SDI, which was calculated as average of N2O fluxes from three different locations , was reduced by 50% compared to furrow irrigation. However, they did not consider the differences in N2O fluxes among different locations around the drip emitter. The current research tried to quantify N2O emissions from sub-regions around the emitter of soil irrigated by SDI with different surface soil wetting proportions. The results indicated that SDI resulted in decreased N2O emission from soils when compared to SI. Furthermore, N2O fluxes from SDI-irrigated soils were generally reduced with a decrease in surface soil wetting proportion, and from the central sub-region to the periphery sub-region. To reduce N2O emissions, it is better to reduce the surface soil wetting proportion and moisture level in topsoil in the sub-regions of SDI irrigated soils. Thus, an ideal or optimal soil wetting pattern should be designed by manipulating SDI technique parameters , with soil moisture high enough to meet crop water requirements as the prerequisite. Balancing the goal of decreased N2O emissions while properly meeting crop water requirements is another issue we should address in future research. It can be concluded that SDI is a prospective irrigation method for N2O emission reduction if it is designed properly, although it accounts for a very small proportion of the irrigation area in China, or even globally (no more than 0.6%) [2O emissions.Irrigation management is an important practice that determines soil moisture distribution characteristics, and therefore Nn et al. ,17 invesh et al.  demonstran 0.6%) . Conside2O emissions [2O emissions should also be examined in field experiments under the conditions including crop cultivation and N fertilization management in the future. These results will be helpful for understanding the influence and mechanisms of water and nitrogen dynamics on crop growth and N2O emissions under SDI irrigation, and for guiding water and fertilizer management practices in agricultural production to realize N2O emission mitigation. Other than soil moisture, nitrogen (N) fertilizer is another important factor that affected Nmissions ,36. The 2O emissions from SDI irrigated soils with different surface soil wetting proportions were investigated in the present research. Results indicated that surface soil wetting patterns caused by SDI significantly affected soil N2O emissions. The wetting process among sub-regions under all SDI wetting patterns resulted in pulse N2O effects; however the magnitudes and occurrence times of pulse N2O fluxes were reduced and lagged with the decrease of surface soil wetting proportions. Generally, peak N2O fluxes from SDI irrigated soils increased exponentially with the increase of the maximum WFPS, and the magnitudes of pulse N2O fluxes were positively correlated with the match between the degree of soil WFPS and the optimal soil moisture level for peak N2O emissions. Compared to SI, N2O emissions from SDI irrigated soils reduced from SDI–1 (surface soil wetting proportion is 39.7%) to SDI–3 soil (surface soil wetting proportion is 1.7%). Furthermore, within a specific SDI wetting pattern, N2O emissions reduced from central sub-regions to periphery sub-regions. The pulse period contributed most to the reduction of N2O emissions between SI and SDI treatments and should be a key period for N2O emission mitigation. These results suggest that reducing the increment of topsoil WFPS or surface soil wetting proportions is a promising strategy for N2O emission reduction. However, further research should address more typical wetting patterns in circumstances in which the soil moisture is high enough to meet crop water requirement as the prerequisite, to explore an ideal or optimal soil wetting pattern by manipulating SDI technique parameters.N


It has been shown to have potential anti-inflammatory activities, but the molecular mechanism has remained unclear. In this study, we found that nuciferine (10 μM) significantly inhibited the lipopolysaccharide (LPS)-induced inflammatory cytokine IL-6 and TNF-α production in RAW 264.7 cells. In addition, the luciferase reporter assay results of different subtypes of the peroxisome proliferator-activated receptor (PPAR) showed that nuciferine dose-dependently activated all the PPAR activities. Specific inhibitors of PPARα and PPARγ significantly abolished the production of inflammatory cytokines as well as IκBα degradation. However, PPARδ inhibitor did not show this effect. Our results suggested a potential molecular mechanism of the anti-inflammatory effects of nuciferine in LPS-induced inflammation, at least in part, by activating PPARα and PPARγ in RAW 264.7 cells.Inflammation is important and has been found to be an underlying cause in many acute and chronic human diseases. Nuciferine, a natural alkaloid containing an aromatic ring, is found in the  Inflammatory responses are widely implicated in vast kinds of acute and chronic human diseases, including cancer, atherosclerosis, and diabetes . MacrophRegulation of the inflammatory response depends on a variety of potential mechanisms, including peroxisome proliferator-activated receptors (PPARs) actions . PPARs aR)-1,2-dimethoxyaporphine; Nuci) is an alkaloid found within the leaves of Nymphaea caerulea and Nelumbo nucifera, which is widely planted in Asia, the Middle East, and some countries in Africa [The natural product nuciferine  were found between control and all the treatment groups, indicating that nuciferine had no direct cytotoxicity, in this cell line. To avoid using a concentration of nuciferine higher than the normal physiological concentration, 10 μM was used in all of the following experiments.To test the effect of nuciferine on the cell viabilities of RAW264.7 cells, 3--2,5-diphenyltetrazolium bromide (MTT) assay was performed in RAW264.7 cells, using different concentrations of nuciferine, ranging from 1 to 50 μM. After treatment for 24 h, cell viabilities were measured, and the results are shown in p < 0.05) both TNFα and IL-6 levels by 679.2% and 472.6%, respectively. Importantly, the nuciferine decreased both these cytokine levels induced by the LPS, in a dose-dependent manner. Meanwhile, gene expression of these cytokines, by RT-qPCR, showed the same trends as the ELISA results  or mRNA level . Similarly, significant negative correlations were found between nuciferine concentration and TNFα protein level  or mRNA level . All these results indicated that nuciferine had a potential anti-inflammatory effect, by reducing inflammatory cytokines production.In order to evaluate whether the nuciferine has potential anti-inflammatory activity in RAW264.7 cells, the cells were pretreated with nuciferine (1~50 µM), for 24 h, before exposure to LPS, and the pro-inflammatory cytokines IL-6 and TNFα were examined . Without results C,D. Pearr = 0.70; p = 0.004, n = 15) and a significant positive correlation of the ratio of nuciferine concentration versus the PPARγ activity . However, there was no significant correlation of the ratio of nuciferine concentration versus the PPARβ/δ activity . Meanwhile, the luciferase reporter assay was carried out using the HEK 293 cells  was purchased from APP-CHEM . Dulbecco’s modified Eagle’s medium-high glucose (DMEM), fetal bovine serum (FBS), 3--2,5-diphenyltetrazolium bromide (MTT), and Lipofectamine 2000 reagent were purchased from Invitrogen . Lipopolysaccharide (LPS), PPARs agonists WY14643, GW501516, rosiglitazone (Rosi) were purchased from Sigma . TRIzol reagent was purchased from Invitrogen . IL-6 and TNF-α Mouse ELISA Kit was obtained from Elabscience Biotechnology Co. Ltd. . Super Script II Rnase H Reverse Transcriptase kit was purchased from Invitrogen .2 and 95% air. RAW264.7 cells were seeded into plates and treated at approximately 80% confluence.Murine macrophage RAW264.7 cells  and human embryonic kidney cells  were cultured with DMEM containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were maintained at 37 °C, in a humidified atmosphere of 5% CO3 cells/well in 96-well plates. After 24 h, cells were treated with different concentrations of nuciferine (0–50 μM), for 24 h, followed by an addition 20 μL MTT solution (5 g/L), to each well, for 4 h. The insoluble formazan product was dissolved in 150 μL/well dimethyl sulfoxide (DMSO), after washing out the supernatant [490 of sample)/A490 of control well.RAW264.7 cells were seeded at a density of 1.5 × 10ernatant . Then, tRAW264.7 cells were grown into 12-well plates, treated with different concentrations of nuciferine  and stimulated with LPS (500 ng/mL). Cell-free supernatants were collected and the levels of pro-inflammatory cytokines, TNFα and IL-6 were measured using ELISA kits, by a determination of the absorbance at 450 nm, according to the manufacturer’s instructions. Standard curves were used to calculate the concentration of TNFα and IL-6 in each sample.5 cells/well without antibiotics. After 24 h at 60% confluence, cells were transfected according to the manufacturer’s instructions. Briefly, PPARs isoforms (PPARα/PPARδ/PPARγ) plasmid (0.9 µg), reporter plasmid PPRE×3-TK-LUC (0.3 µg) and β-gal (0.1 µg) were transfected into the cells, using Lipofectamine 2000 reagents (1:1), for 4 h. Since transfection efficiency is typically low in RAW264.7 cells, more Lipofectamine 2000 was needed (1:2.5) and the transfection time was extended to 24 h. The medium was replaced with a complete media containing DMSO, nuciferine, or PPARs agonists, for 24 h. The cells were harvested and lysed to measure the luciferase activities using a luciferase assay kit, according to the manufacturer’s instructions. The β-gal was transfected to normalize the transfection efficiency [HEK293T cells and RAW264.7 cells were plated into 12-well plates at 4 × 10ficiency .5 cells/well. Total RNA was isolated using TRIzol reagent and reverse transcribed into cDNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed as described by Yang et al. [Gapdh) was used as an internal control. Ct values of the sample were calculated, and the mRNA levels were analyzed by 2−ΔΔCt method and normalized to Gapdh [Cells were cultured into 12-well plate with a density of 4 × 10g et al. . Glycerato Gapdh . The priRAW264.7 Cell lysates were prepared using a lysis buffer containing 0.1% Triton X-100 and proteinase inhibitors . Protein concentrations were determined using the BCA protein assay kit . Western blot was performed as described by Yang et al. . After bt test and ANOVA followed by Tukey’s post hoc test were used to analyze the significant difference between two or more groups, respectively. The rank-based test methods were employed when data were not in a normal distribution or the variances were not homogeneous. All the results were representative of at least three independent experiments.Quantitative data are expressed as mean ± SEM using SPSS 18.0 . Student


The SNAREs Vti1a/1b are implicated in regulated secretion, but their role relative to canonical exocytic SNAREs remains elusive. Here, we show that synaptic vesicle and dense-core vesicle (DCV) secretion is indeed severely impaired in Vti1a/b-deficient neurons. The synaptic levels of proteins that mediate secretion were reduced, down to 50% for the exocytic SNARE SNAP25. The delivery of SNAP25 and DCV-cargo into axons was decreased and these molecules accumulated in the Golgi. These defects were rescued by either Vti1a or Vti1b expression. Distended Golgi cisternae and clear vacuoles were observed in Vti1a/b-deficient neurons. The normal non-homogeneous distribution of DCV-cargo inside the Golgi was lost. Cargo trafficking out of, but not into the Golgi, was impaired. Finally, retrograde Cholera Toxin trafficking, but not Sortilin/Sorcs1 distribution, was compromised. We conclude that Vti1a/b support regulated secretion by sorting secretory cargo and synaptic secretion machinery components at the Golgi. Vti1a and Vti1b are SNARE proteins implicated in regulated secretion. Here the authors show that Vti1a and Vti1b control protein sorting at the Golgi, and targeting to the axon, and thereby indirectly control regulated secretion at the synapse. Immature DCVs are proposed to undergo initial fusion reactions, homotypic fusion9 or heterotypic fusion to endosomes10, before they are fusion-competent. Fusion requires formation of SNARE complexes consisting of four SNARE protein domains . In addition to the canonical exocytic SNARE proteins , other R- and Q-SNAREs are implicated in regulated secretion. However, how these additional SNAREs regulate secretion in relation to the canonical exocytic SNAREs is still poorly understood.Regulated secretion of signaling molecules from synaptic vesicles (SVs) and dense-core vesicles (DCVs) is the primary means of neuronal communication. SVs store neurotransmitters and are locally recycled after exocytosis14. These proteins function in endo-lysosomal trafficking19 and combined gene inactivation in mice triggers neurodegeneration and perinatal death12. Vti1a knockdown results in reduced spontaneous SV fusion39. However, it is unclear which domain Vti1a would contribute to exocytic SNARE complexes because SNAP25 supplies the Qb-SNARE domain for spontaneous SV fusion23. Vti1a knock-out chromaffin show reduced exocytosis by unknown pathways that result in decreased numbers of secretory granules24. Hence, it is possible that, also in neurons, dysregulation of Vti1a/b-dependent upstream pathways contributes to downstream secretion defects.In this context, the Qb-SNARE Vti (VPS10 interacting) proteins are particularly enigmatic. Two Vti proteins are ubiquitously expressed in mammals, Vti1a and Vti1bHere, Vti1a/b-null neurons were used to test this hypothesis. We found that the number of DCVs and synapses, and the synaptic levels of proteins that mediate secretion were all reduced. The secretion efficiency of SVs and DCVs was severely impaired. The delivery of DCV-cargo and SNAP25 into axons was reduced. DCV-cargo and SNAP25 accumulated in the Golgi. These phenotypes were rescued by expression of either Vti1a or Vti1b. Furthermore, Golgi cisternae were distended and the export of cargo from the Golgi was impaired. We conclude that Vti1a/b regulate secretion by sorting secretory cargo and proteins of the secretion machinery out of the Golgi.12, with double heterozygotes (DHZ) as controls  mice26. At DIV-14, expression of both markers was punctate  were quantified by staining for endogenous Synaptophysin-1 Fig. , and DCVate Fig. . In DKO ate Fig. , which rate Fig.  and 15% ate Fig.  reductioate Fig. . Furtherate Fig.  and 50% ate Fig. . The endate Fig. , withoutate Fig. . Expressate Fig. , except ate Fig. .At the ultrastructure level, random cross sections of synapses contained similar numbers of SVs, docked SVs per synapse, active zone length and postsynaptic density length between DHZ and DKO Fig. . HoweverThese results show that Vti1a/b deficiency results in in vitro cell death, and that the surviving neurons are smaller and have fewer synapses and DCVs. Furthermore, expression of either Vti1a or Vti1b rescues virtually all these phenotypes equally well.27 (SypHy) and FM4-64-labeled SVs  stimulation was decreased by 80–90% Fig. . The amp26, triggered similar calcium transients in all groups , Munc13-1 46.1%), Bassoon (35.5%), Synaptotagmin-1 (29.8%), and RIM1/2 (19.2%)  co-localized with Vti1a and Vti1b Fig. . Vti1a aBecause of the preferential localization of Vti1a/b to the Golgi, we examined the structure of this organelle in DKO neurons. At DIV-14, the total areas stained for the cis-Golgi marker GM130 and the trans-Golgi marker TGN46 were reduced in DKO neurons Fig. , but theWe subsequently investigated a potential relationship between Golgi defects and the reduced cargo delivery to axons at DIV-5. At DIV-5, the areas stained by GM130, ManII-EGFP, and TGN46 were already smaller in DKO neurons  was co-expressed in DIV-5 neurons with the ER-resident protein KDEL fused to Streptavidin. The interaction between Streptavidin and SBP traps cargo at the ER29  within minutes  co-localization of Vti proteins with endo-lysosomal markers. We also found that Vti proteins co-localize at the Golgi and that many Vti1a/b-null phenotypes in neurons were fully rescued by the expression of either Vti1a or Vti1b, indicative of redundant functions. Furthermore, the fact that DKO mice show neurodegeneration and die at birth, while single null mutants are viable and fertile36 supports shared functions for these proteins. Hence, Vti1a and Vti1b support many cellular functions in neurons in an equally efficient and largely redundant manner. Such redundancy may have remained unnoticed because most of the previous studies selectively addressed single Vti proteins and/or used systems where only a single-Vti protein may be expressed. Alternatively, redundancy may be specific for neurons, which is supported by the fact that neurons are the most affected cell type in the DKO mice12. The reduced DCV pool and impaired CTB delivery to the Golgi were the only two phenotypes more efficiently rescued by Vti1a than Vti1b. This aligns with the decreased number of secretory granules in Vti1a KO chromaffin cells24 and the Vti1a-dependent transport of Shiga toxin to the Golgi in cell lines16. Hence, although few aspects of intracellular trafficking are more efficiently supported by Vti1a, Vti1a and Vti1b generally support cellular functions in a largely redundant manner, at least in neurons.Vti1a and Vti1b are the two mammalian orthologs of the single-yeast 37 and local roles in endosomal sorting of SVs38 and spontaneous SV fusion39 have been proposed. This latter proposal was supported by the spontaneous fusion of Vti1a-labeled acidic organelles (which were distinct from Syntaxin6- or transferrin receptor-labeled organelles) and by a reduced spontaneous SV fusion upon single Vti1a or combined Vti1a/VAMP7 knockdown39. We observed that SV and DCV secretion were severely impaired in Vti1a/b-deficient neurons. Because SNAP25 supplies the Qb domain in the SNARE complex that drives SV exocytosis, also spontaneous23, it seems improbable that the Qb-SNAREs Vti1a/b have a direct role in SV fusion with the plasma membrane. Our data demonstrates that Vti1a/b maintain normal levels of, at least, SNAP25, Munc13-1, RIM, Bassoon, and Synaptotagmin-1 and, to a lesser extent, Syntaxin-1, Synaptobrevin-2, and Munc18-1, and that at least SNAP25 accumulates at the Golgi. Deficiency for most of these proteins inhibits SV fusion41 and DCV fusion42, and haploinsufficiency also affects synaptic transmission for at least, SNAP25 or Munc18-144. Hence, the combined reduction of the levels of these proteins provides a plausible explanation for the impaired secretion in the absence of Vti1a/b. Although our data cannot exclude local synaptic functions for Vti1a/b, we conclude that Vti1a/b support synaptic secretion by sorting and targeting components of the secretion machinery to synapses.Vti1a is detected in SV fractions2, and does not depend on Vti1a/b. Furthermore, given the reduced staining of synaptic proteins, SVs most likely contain a reduced number of native proteins per vesicle in DKO neurons. This provides a plausible explanation for the impaired synaptic transmission in Vti1a/b neurons.Fewer DCVs were observed in synaptic micrographs of DKO neurons and staining for DCV-cargo was reduced, whereas DCV cargo loading per vesicle was unaltered. This indicates that DKO neurons express fewer DCVs with probably unaltered cargo composition. In contrast, the number of SVs per synapse was unaffected, while staining intensity for synaptic markers was decreased in DKO synapses. This suggests that, unlike DCV biogenesis, SV biogenesis is unaffected by the loss of Vti1a/b, despite the reduced levels of synaptic proteins. We conclude that SV biogenesis is coupled to the rate of exocytosis, as suggested9, arguing for the first scenario. However, the somatic distribution of DCVs and the cargo content of DCVs were normal in Vti1a/b-deficient neurons, suggesting unaffected maturation. A different Qb-SNARE may support vesicle maturation or, alternatively, this process may not be essential in neurons. In contrast, we observed independent lines of evidence for the second scenario, impaired Golgi export. The Golgi was smaller and cisternae were abnormal, the normal non-homogeneous distribution of DCV-cargo inside the Golgi was lost, cargo accumulated at the Golgi and the transit time of labeled cargo through this organelle was abnormally long. In addition, the export of labeled cargo was also delayed in astrocytes, indicating that Vti1a/b do not only support this function in neurons. Therefore, we conclude that Vti1a/b are required for normal protein export from the Golgi.The influx of DCV-cargo and proteins of the secretion machinery into the axon was decreased in DKO neurons, but co-localization of these proteins with lysosomes or their cellular stability were unaltered. Therefore, the reduced levels of DCVs and proteins that drive secretion are most likely explained by decreased axonal influx, not by aberrant protein turnover. The two main cellular pathways that can explain the decreased influx into axons are impaired maturation of transport vesicles (by homotypic or heterotypic fusion) or impaired protein export from the Golgi. The Vti partner Syntaxin-6 mediates homotypic fusion of secretory organelles ex vivoVti1a-null chromaffin cells24 and the accumulation of tumor necrosis factor-α in a Golgi-like compartment in macrophages after Vti1b inhibition45. Our study shows that targeting of cargo presumably not sorted to the regulated secretory pathway, like SNAP25, GPI, PSD95, and TrKB receptors, was also impaired in DKO neurons. This is in line with the impaired delivery to the plasma membrane of Ca2+ channels in Vti1a-null chromaffin cells24 and Kv4 K+ channels in HeLa cells deficient for Vti1a or the Vti SNARE partner VAMP746, and indicates that the exit of many proteins from the Golgi relies on Vti1a/b.The accumulation of DCV-cargo in the Golgi and its abnormally long transit time are consistent with the reduced number of secretory granules in 12. Because neuronal death was already abundant at DIV-4, when regulated secretion is minimal21, and because abolishing regulated secretion perse does not trigger neuronal death47, the degeneration observed in DKO neurons is probably not a direct consequence of impaired exocytosis. However, we cannot exclude that the combined loss of (many) membrane proteins contributes to the degeneration observed. Despite the accelerated cell death of DKO neurons, many cellular functions were normal. The distribution of synapses and DCVs was unaffected, DCV trafficking speed (around 1 µm/s) was in the range of reported values48; and SV endocytosis, calcium influx and efflux and vesicular acidification were similar to DHZ neurons, confirming that Vti1a/b DKO neurons can cope with energy-demanding tasks. Furthermore, protein stability, mitochondrial morphology and ER-to-Golgi cargo trafficking were unaffected. This is in line with previous observations that many intracellular trafficking pathways, including recycling of transferrin receptors, delivery of endocytosed cargo to lysosomes and autophagy, were fully functional in Vti1a/b DKO fibroblasts12. Golgi abnormalities in the absence of Vti1a/b were also observed in acute brain slices, indicating that this is not a feature unique for cultured neurons. Furthermore, the levels of synaptic proteins and SV fusion were rescued by expression of Vti1a or Vti1b after most DKO neurons died, which confirms that these features are not intrinsic of the surviving neurons. Hence, we conclude that loss of Vti1a/b function does not trigger generalized cellular dysfunctions, but selectively affects regulated secretion by impaired Golgi export of relevant proteins, which is not a unique feature for surviving neurons or culture conditions.Neurons degenerate in the absence of Vti1a/b49. ER-Golgi trafficking was normal in Vt1a/b-deficient neurons and astrocytes, in line with the fact that other Qb-SNAREs mediate this anterograde pathway51. In contrast, retrograde transport from the plasma membrane to the Golgi was impaired in Vti1a/b-deficient neurons, consistent with the decrease in Shiga toxin transport in cell lines upon interference with Vti1a-function using antibodies16. The preferential localization of Vti1a/b to the Golgi aligns with a role in retrograde transport to this organelle. Vti1b localized to the Golgi in neurons to a larger extent than what was reported in fibroblast cell lines52, which may indicate cell type differences. Vti1a/b deficiency had a larger impact on retrograde transport at DIV-14 than at DIV-5. This can generally be explained by a progressive nature of this defect: components that regulate this trafficking probably become progressively missorted over time. Alternatively, different Qb-SNARE may be involved in similar pathways during different developmental stages21, or synaptic activity influences CTB retrograde trafficking, as suggested30. Expression of either Vti1a or Vti1b in DKO neurons increased CTB delivery to the Golgi, but only Vti1a fully rescued it. Retrograde transport of Shiga toxin to the Golgi depends on EpsinR53, a Vti1b interactor54, which confirms the relevance of Vti1b in plasma membrane-to-Golgi transport. In addition to plasma membrane–Golgi transport, additional retrograde transport routes may be affected in DKO neurons. Indeed, Vti1a mediates late endosome-to-Golgi transport17, also in yeast55. Defective transport of sorting molecules between endo-lysosomal organelles and the Golgi may also help to explain Golgi export defects in Vti1a/b DKO neurons, and would fit with the role of endo-lysosomal proteins in sorting of regulated secretory cargo57.Molecules are delivered to the Golgi anterogradely from the ER, or retrogradely from the plasma membrane or intracellular organelles58. Second, disrupting either the Conserved Oligomeric Golgi (COG) or the GARP tethering complexes, which function in recycling to or within the Golgi, resulted in distended Golgi cisternae and vacuolization61. These similarities suggest that Vti1a/b may work together with one or both these complexes to regulate transport to or within the Golgi and that impaired delivery of molecules that control protein sorting may explain the cellular phenotypes described in our study -complex subunit Vps54 in cell lines resulted in inefficient transit of GPI from the Golgi to the cell surfaceudy Fig. . Interes34. In mammals, the VPS10 receptors Sortilin and Sorcs1 bind and sort regulated cargo39, and would provide a plausible link between retrograde trafficking defects and other cellular phenotypes observed in Vti1a/b DKO neurons. However, the levels and distribution of these receptors were unaltered in DKO neurons, and binding to mammalian Vti1a or Vti1b was not detected. Additional molecules have been implicated in protein sorting at the Golgi, including calcium transporters64, proton pumps65, HID-166, CAB4567, and lipids69. Cytosolic proteins, such as actin regulators64, adapter complexes56, enzymes70, and Arf members72, also contribute to Golgi sorting, but the correct localization of these soluble proteins probably does not rely directly on Vti1a/b-dependent membrane trafficking. An overarching model for protein sorting at the Golgi is lacking and the link between molecules that function in this process and Vti1a/b remains unclear. However, the current study clearly indicates that Vti1a/b function is central to coordinate Golgi import/export, with major downstream consequences if this aspect is not functional.In yeast, Vti1p binds to the sorting receptor Vps10 and a Vti1p mutation causes cargo missorting36. Embryonic day (E) 18.5 embryos were obtained by cesarean section of pregnant females from timed mating of Vti1a± and Vti1b ∓ −/ + mice, or vice versa. Mouse hippocampi were dissected from these embryos in HBSS (Sigma), digested in 0.25% trypsin (Life technologies) for 15 min at 37 °C and dissociated with fire-polished Pasteur pipettes. Dissociated neurons were resuspended in Neurobasal supplemented with 2% B-27, 18 mM HEPES, 0.25% Glutamax, 0.1% penicillin/streptomycin (Life technologies), and plated on pre-grown glia cells . For astrocyte micro-islands, 1500 or 4000 neurons/well for DHZ and DKO, respectively, were plated. A higher density was used for DKO to compensate for the neuronal death. DHZ were used as controls due to the low probability to obtain DKO and double-WT mice within the same litter. Neurons were maintained at 37 °C, 5% CO2 for 13–15 days (referred as DIV-14) or 4–6 days (referred as DIV-5).Rat and mouse astrocytes were cultured from P1-2 pup cortices. For functional experiments, astrocytes were plated in T75 flasks, grown in DMEM with 10% FCS, 1% NEAA, 1% penicillin/streptomycin (Life Technologies) at 37 °C, 5% CO2 till confluent, and subsequently replated on glass coverslips and grown for 1 week. For micro-islands, astrocytes were prepared by plating 6000 rat glia cells per agarose-coated glass coverslip stamped with 0.1 mg/ml poly-d-lysine (Sigma) and 0.7 mg/ml rat tail collagen (BD Biosciences). Animals were housed and bred according to institutional and Dutch guidelines.Vti1a/1b null mice, generated by replacing a 6.5 Kb fragment in Vti1a exon 6 and a 7 Kb fragment in Vti1b exon 4 with neomycin resistance cassettes, have been described before36. NaVII/III-mCherry was engineered by substituting YFP by mCherry in NaVII/III-YFP . ManII-ECFP was made by substituting GFP by ECFP in ManII-GFP . The above plasmids were cloned into pLenti vectors under the human Synapsin promoter and viral particles were delivered to neurons at DIV-1 or DIV-10. For RUSH experiments, CMV driven Streptavidin-KDEL/SBP-EGFP-NPY was engineered substituting GPI from Streptavidin-KDEL/SBP-EGFP-GPI . Standard calcium phosphate precipitation was used to deliver these plasmids, in combination with ManII-EGFP. Cells were analyzed 20–28 h after transfection.Vti1a, Vti1b, NPY-pHluorin, NPY-mCherry, Synapsin-ECFP, and Synaptophysin-pHluorin plasmids have been describedFor immunoprecipitation, Vti1a and 1b were fused to Myc tags, and SorCS1  and Sortilin  were fused to Flag tags. These plasmids were expressed under CMV promoters and transfected into HEK293T cells.2HPO4, 1.8 KH2PO4; pH = 7.4). Coverslips were mounted in Mowiol (Sigma) and Z-stacks were obtained on a confocal microscope (Nikon Eclipse Ti) equipped with ×10 (NA = 0.45) and ×63 oil (NA = 1.40) objectives controlled by NisElements 4.30 software. For retrograde labeling experiments, neurons were incubated at 37 °C with CTB-A488  for 15 min and fixed 30 min or 2 h after incubation. For protein stability, neurons were incubated with cycloheximide  in 0.1% DMSO, or 0.1% DMSO alone, for 24 h before fixation.Neurons, fixed in 3.7% paraformaldehyde (EMS) for 20 min, were permeabilized with 0.5% Triton-X (Fisher Scientific) for 5 min and blocked with 2% normal goat serum (Life Technologies) and 0.1% Triton-X for 20 min. Incubation with primary and secondary antibodies was done at room temperature (RT) for 2 or 1 h, respectively. All solutions were in PBS , MAP2 , Munc18-1 , SNAP25 , Synaptobrevin-2 , Syntaxin-1 , Synaptophysin-1 , Munc13-1 , Synaptotagmin-1 , RIM , Bassoon , GM130 , TGN46 , SMI312 , VGLUT-1 , VGAT , Lamp1 , BDNF , TrKB , PSD95 , Rab5 rabbit , and Rab5 mouse . Secondary AlexaFluor-conjugated antibodies .Primary antibodies used for immunocytochemistry: Vti1a , Vti1b  were obtained and deparaffinized. Deparaffinized sections were renatured by microwaving for 5 min in boiling 0.1 M citrate buffer (pH 6) and incubated with 1% normal goat serum in PBS for 1 h. Renatured sections were incubated with primary mouse anti-GM130 antibody  overnight at 4 °C, followed by Cy3-cojugated anti-mouse antibodies  for 1 h at RT. Nuclei were stained in Hoechst dye .2, 2.5 KCl, 119 NaCl, 2 MgCl2, 20 Glucose, 25 HEPES, pH 7.4) and recorded at RT using a Zeiss AxioObserver.Z1 (SV and DCV fusion) or at 37 °C (FRAP and RUSH) using a Nikon Eclipse Ti microscope. Microscopes were equipped with laser-based illumination , appropriate filter sets, ×40 oil (NA = 1.3) or ×63 oil (NA = 1.4) objectives and an EM charge-coupled device camera . Time-lapse recordings were acquired using Axiovision 4.8 or NisElements 4.30. The acquisition frequency was 2 Hz for DCV fusion, FRAP and calcium imaging; 0.6 Hz for dual color (SypHy/FM4-64) SV fusion and 1 Hz for single (SypHy) SV fusion rescued at DIV10. HFS was applied using parallel platinum electrodes delivering 30 mA, 1 ms pulses controlled by a Master-8 (AMPI) and a stimulus generator . Intravesicular pH was neutralized by gravity flow application of modified Tyrode’s solution (50 mM NaCl replaced by 50 mM NH4Cl). For FRAP experiments, laser intensity and pulse duration for bleaching were optimized to reach >90% fluorescence decrease of NPY-mCherry or SNAP25-EGFP at the axon initial segment (labeled using NaVII/III-mCherry or -EYFP). For calcium imaging, neurons were incubated with 2 µM Fluo5F AM (Molecular Probes) in supplemented Neurobasal at 37 °C, 5% CO2 for 10 min. For RUSH experiments29, neurons were imaged in supplemented Neurobasal. Cycloheximide  and Biotin  were added at recording time-0. FM4-64  was dissolved in Tyrode’s containing 60 mM K+ and incubated for one minute, followed by a 10-min wash, before imaging.Coverslips were placed in imaging chambers perfused with Tyrode’s buffer  over initial fluorescence . For SV fusion, 4×4 pixel ROIs were placed on all synapses, identified as non-mobile SypHy puncta with a ΔF/F0 > 3 standard deviation (sd) upon NH4+ superfusion. For DCV fusion, 3×3 pixel ROIs were manually placed on all NPY-pHluorin puncta that appeared upon HFS stimulation. Only when ΔF/F0 > 3 sd, ROIs were counted as DCV fusion events. The total DCV pool was quantified from the maximum projection during NH4+ superfusion. SynD was used for DCV puncta detection. The detected puncta may contain several DCVs due to resolution limitations and, hence, pool underestimation was avoided by applying puncta intensity correction. Intensity correction considers that the modal puncta intensity per neuron corresponds to the intensity of individual DCVs that fuse, as demonstrated25. For synaptic DCV events, binary masks were built in ImageJ  based on Synapsin-ECFP. DCV fusion events with >50% pixels within the binary mask were considered synaptic. For FRAP experiments, the number and speed of NPY-mCherry or SNAP25-EGFP puncta that entered the axon from the soma were calculated using kymographs (ImageJ). For visualization purposes, brightness, and contrast of representative examples was adjusted in a linear and equal fashion between groups.A custom-written algorithm in MATLAB (Mathworks), SynD2, 5.0 mM EDTA (pH 8), 100 mM NaCl) 48 h after transfection. Samples were incubated with antibodies against Flag  or Myc tags , and protein-A agarose beads (Vector Laboraties). Beads were collected upon centrifugation  and washes with buffers (50 mM Tris-HCl (pH 7.5), 0.1% TX-100, 1.5 mM MgCl2, 5.0 mM EDTA (pH 8)) containing 100 or 200 mM NaCl. Finally, beads were dried using an insulin needle, dissolved in Laemmli Sample buffer, boiled, and analyzed by western blotting.For expression of Vti1a and Vti1b, DIV-14 neurons (cultured in absence of glia) were washed in PBS, homogenized in Laemmli buffer  and analyzed by western blot. For immunoprecipitations (IPs), HEK293T cells were transfected with Flag- and Myc-tagged proteins and lysed in IP buffer (50 mM Tris-HCl (pH 7.5), 1% TX-100, 1.5 mM MgClFor Western blots, samples were separated on 10% SDS-polyacrylamide gels and transferred to Nitrocellulose or PVDF membranes. Blots were blocked in 2% milk and 0.5% bovine serum albumin (BSA) for 1 h at 4 °C and incubated with primary and secondary antibodies at 4 °C overnight and 1 h at RT, respectively. Blots were scanned using a Fuji Film FLA 5000. Blocking and antibody solutions were prepared in PBS containing 0. 1% Tween-20.4/1% KRu(CN)6. After dehydration through a series of increasing ethanol concentrations, cells were embedded in Epon and polymerized for 48 h at 60 °C. Ultrathin sections (80 nm) were cut parallel to the cell monolayer, collected on single-slot formvar-coated copper grids, and stained in uranyl acetate and lead citrate in Ultra stainer LEICA EM AC20. Neuronal sections were randomly selected at low magnification and imaged using a JEOL1010 transmission electron microscope at 60 kV.Neurons were fixed for 90 min at RT with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. Cells were washed and postfixed for 1 h at RT with 1% OsO+‐gluconic acid, 10 NaCl, 4.6 MgCl2, 4 K2‐ATP, 15 creatine phosphate, 1 EGTA, and 10 units/mL phosphocreatine kinase (pH 7.30). External solution contained in mM: 10 HEPES, 10 glucose, 140 NaCl, 2.4 KCl, 4 MgCl2, and 2 CaCl2 . Inhibitory neurons were identified and excluded based on the decay of postsynaptic currents. Recordings were acquired with a MultiClamp 700B amplifier, Digidata 1440 A, and pCLAMP 10.3 software (Molecular Devices). Only cells with an access resistance < 15 MΩ (80% compensated) and leak current of <300 pA were included. EPSCs were elicited by a 0.5 ms depolarization to 30 mV. RRP size was assessed by hypertonic sucrose application74 (500 mM for 7 s), using a piezo-controlled barrel application system  and fitted as reported75. Offline analysis was performed using custom‐written software routines in Matlab R2016b (Mathworks)In all figures, stimulation artifacts have been blanked out.Whole‐cell voltage‐clamp recordings (Vm = −70 mV) were performed at RT with borosilicate glass pipettes (2–5 MΩ) filled with (in mM) 125 Kt-test or ANOVA (Tukey as post hoc). Otherwise, non-parametric tests were used: Mann–Whitney U or Kruskal–Wallis test (Holm as post hoc). R was used as software. No predetermined sample size or randomization were used. No data was excluded. Data was analyzed blindly.Shapiro and Levene’s were used to test distribution normality and homogeneity of variances, respectively. When assumptions of normality or homogeneity of variances were met, parametric tests were used: The custom code used for data analysis during the current study is available from the corresponding author on reasonable request.The data sets generated and analyzed during the current study are available from the corresponding author on reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


The click polymerization completed in merely 4 h to produce the desired MIP–NSHs, which were characterized by FTIR, SEM, DLS, and BET, respectively. The reaction conditions for adsorption capacity and selectivity towards hypericin were optimized, and the MIP–NSHs synthesized under the optimized conditions showed a high adsorption capacity (Q = 6.03 μmol·g−1) towards hypericin. The imprinting factors of MIP–NSHs towards hypericin, protohypericin, and emodin were 2.44, 2.88, and 2.10, respectively.To fabricate molecularly imprinted polymer nanospheres via click reaction, five different clickable compounds were synthesized and two types of click reactions  were explored. It was found that molecularly imprinted polymer nanospheres could be successfully synthesized via thiol-yne click reaction using 3,5-diethynyl-pyridine ( As an important type of artificial receptor, molecularly imprinted polymers (MIPs) ,2, propoThe most common physical form of MIPs is polymer monolith obtained via bulk polymerization, which usually produces irregular polymeric particles in a range of 5–100 μm in low yield (less than 50%) ,14 throuClick reaction, introduced by Sharpless in 2001 , has beeHypericum perforatum (St John’s wort) by multistep extraction, therefore, the development and application of hypericin imprinted polymer can speed up and improve the isolation process or can be used in a sensor for hypercin monitoring in biological samples [1) as the functional monomer, trimethylolpropane tris(3-mercaptopropionate)  as the crosslinker in the presence of hypericin (see Q = 6.03 μmol·g−1) and fair selectivity towards hypericin. This work presents a straightforward method to fabricate molecularly imprinted nanospheres via thiol-yne click reaction.Previously, with ultrasonic assistance, our group developed a one-step approach to synthesize polymeric nanospheres via click reaction between azide and alkyne ,31 in th samples . In thisicin see . The thi1H NMR , 2,6-dibromopyridine (98%) were purchased from Daejeon Fung Teng Co., Ltd. . The chemical 2-methyl-3-butyn-2-ol (98%) was purchased from J & K . Copper(I) Iodide (98%), trimethylolpropane (>98%), pentaerythritol (98%), bis(triphenylphosphine)palladium(II) chloride (Pd 15.2%), and sodium methylate (97%) were purchased from Aladdin . Trimethylsilylacetylene (98%) was purchased from Energy Chemical . Benzoin dimethyl ether (DMPA) (98%) was purchased from Heowns . Sodium azide (99%) was purchased from Xiya Reagent Co., Ltd. . Potassium hydroxide, acetonitrile, acetone, DMSO were purchased from Kelon Chemical Co., Ltd. . Toluene, triethylamine, methanol, sulfuric acid (98%), and all the other reagents were purchased from Tianjin Bodi Chemical Industry Co, Ltd. . Emodin was purchased from Tianfeng Biological Technology Co, Ltd. . Protohypericin (Protohyp) and hypericin (Hyp) were synthesized according to the procedures developed in our lab , and cha1H in DMSO-d6, CDCl3, or MeOH-d4. The residual signals from DMSO-d6 (1H: δ 2.50 ppm), CDCl3 (1H: δ 7.26 ppm), or MeOH-d4 (1H: δ 3.31 ppm) were used as internal standards. Dynamic light scattering (DLS) analysis was performed on a DelsaTM Nano system . The surface area and the porosity of the prepared polymeric nanospheres were determined using nitrogen physisorption based on the Brunauer-Emmet-Teller (BET) method . Samples were vacuum-degassed at 50 °C for 9 h before the adsorption experiments. Field emission scanning electron microscopy (FESEM) images were obtained with a Hitachi-S4800 instrument  operating at 10 kV. High performance liquid chromatography (HPLC) was performed with C18 reversed-phase column  at 25 °C. The mobile phase consisted of 50% acetonitrile, 50% of the mixture of ammonium acetate-acetic acid buffer , and methanol ; detection wavelength: 590 nm; flow rate: 0.4 mL/min; injection volume: 10 μL. The retention time of hypericin under the given chromatographic system is 15.21 min.NMR spectra were recorded on a Bruker 500 MHz Spectrometer  with working frequencies of 500 MHz for 1) and 2,6-diethynyl-pyridine (2) as the monomers. Three crosslinkers of 1,3-bis(azidoacetoxy)-2-azidoacetoxymethyl-2-ethylpropane , 2,2-bis((2-azidoacetoxy)methyl)propane-1,3-diyl bis(2-azidoacetate) , and trimethylolpropane tris(3-mercaptopropionate)  were chosen for azide-alkyne or thiol-yne click reactions in this work  were prepared as follows. To a quartz tube, 0.1 mmol functional monomer, 0.1 mmol cross-linker, 0.031 mmol DMPA, 2 mL acetone were added, and the mixture was subjected to ultrasonication for 10 min, followed by adding 5 μmol template. The click polymerization was performed for 4 h under UV light at a wavelength of 350 nm at ambient temperature under NThe extraction procedure: The nanospheres were extracted with a soxhlet extractor using 10% acetic acid in acetone for 48 h, thereafter the nanoshperes were ultrasonicated in 5 mL 20% acetic acid in acetone for 20 min and then centrifuged; the resulting supernatant was monitiored using HPLC. The ultrasonication step was repeated until no hypericin could be detected in the supernatant. Control polymer nanospheres (NIP–NSs) were prepared under identical conditions but in the absence of hypericin. Q, μmol·g−1) was calculated according to Equation 1:C0 and Ce represent the initial and equilibrium concentrations of the test molecule in acetone (μM), respectively, V (L) is the volume of the solution, and W (g) is the dry weight of the nanospheres.To determine the static adsorption capacity, 5 mg of the nanospheres (MIP–NSHs or NIP–NSs) was placed into a 10 mL plastic centrifuge tube and mixed with 5 mL of the test molecule acetone solution (10.0 μM) to allow adsorption at 25 °C for 24 h. The concentration of the test molecule in the supernatant was measured with HPLC using a calibration curve see . The adsQs) towards hypericin, is calculated according to Equation 2:Q1 and Q2 are the static adsorption capacity of MIP–NSHs and NIP–NSs (μmol·g−1) towards hypericin, respectively.The neat adsorption capacity of MIP–NSHs over that of NIP–NSs, defined as the specific adsorption capacity of MIP–NSHs (5.0 mg of MIP–NSHs (or NIP–NSs) were mixed with a hypericin acetone solution  in a 10-mL centrifuge tube. The tube was sealed and shaken in the dark at 25 °C for different time intervals . The concentrations of hypericin in the supernatant were determined by HPLC. The respective adsorptions were then calculated according to Equation 1.Six portions of 5.0 mg polymers were weighed into plastic centrifuge tubes and mixed with 5 mL of hypericin acetone solution with different concentrations , respectively. The mixtures were shaken for 8 h at 25 °C, and the concentrations of hypericin in the supernatants were determined by HPLC. The respective adsorptions were then calculated according to Equation 1. The isotherm adsorption curve was plotted based on the concentrations of hypericin in supernatants versus the amount of hypericin bound to MIP–NSHs.SF) and “imprinting factor” (IF) [D is distribution (L·g−1), −1), −1), −1), −1), The binding selectivity of MIP–NSHs and NIP–NSs was evaluated by following the method reported . Brieflyor” (IF) , which aThe reusability of MIP–NSHs was evaluated by an adsorption–extraction cycle experiment. One adsorption–extraction cycle consisted of loading the template and reaching equilibrium adsorption, followed by the extraction of the template. For the adsorption, 5 mg of the MIP–NSHs were incubated with 5 mL of hypericin solution (12.5 μM in acetone) at 25 °C. After incubation under continuous shaking for 8 h, the concentration of hypericin in the supernatant was measured by HPLC. The adsorption capacity was calculated according to Equation 1. The MIP–NSHs were subjected to the extraction procedure mentioned above, and then used for the next cycle. 1 + 3 or 4 and 2 + 3 or 4) initiated by Cu(PPh3)3Br [1 + 3 (Qs = 1.91 μmol·g−1) among the four polymers, as shown in Qs from 2 + 3 3Br . Howeverr [1 + 3 , P1 gaveom 2 + 3 , P2 was 4 instead of tri-azide 3 as the crosslinker did not benefit the adsorption of the resulting MIP–NSHs. For example, the Qs of P3 synthesized from 1 + 4 was much lower than that of P1. This might be because the polymer formed with 4 as crosslinker was denser than that with 3 as crosslinker, which make the removal and re-binding of hypericin from the polymer more difficult, and, as a result, lowered the adsorption of MIP–NSHs drastically. Thus, a tri-thiol crosslinker 5 was synthesized and combined with 1 to fabricate MIP–NSHs via thiol-yne click reaction . Moreover, the absorption band of C–O on hypericin at 1230 cm−1 [To confirm that the thiol-yne click polymerization took place between ups on 1  and S–H 230 cm−1  was foun1 + 5 were characterized with FESEM and DLS analyses. The results are shown in 1 + 5.The polymers obtained via click polymerization between 3·g−1, respectively; which are much larger than those of NIP–NSs, indicating that the former has a porous structure, and the latter a compact one. The average pore diameter, surface area and pore volume of the MIP–NSHs were roughly 13, 2.5 and 8.4 times those of the NIP–NSs, respectively. The average pore diameter, surface area and pore volume of the MIP–NSHs and NIP–NSs fabricated in this work were characterized with BET analysis. The results are summarized in Qs were screened and optimized; this included photoinitiator concentration, solvent composition, hypericin concentration, the equivalent ratio of 1 to 5, etc.In order to obtain MIP–NSHs with the most optimal imprinting effect towards hypericin, the preparation conditions that affected Qs was investigated by varying the concentration of DMPA in a range of 2.0–16 mg·mL−1. As shown in −1, Qs increased dramatically from 0.65 to 2.71 μmol·g−1. However, further increase in the concentration led to the opposite effect with Qs decreasing to 0.57 μmol·g−1 when the concentration was increased to 16 mg·mL−1. Hence, 4 mg·mL−1 was used as the optimal concentration of the photoinitiator for subsequent studies.As previously mentioned, DMPA was used as the photoinitiator in this work, which generated active free radicals upon exposure to UV light and induced monomer and crosslinker click polymerization. The concentration of DMPA affected not only the rate of polymerization, but also the imprinting effect of the resulting imprinted polymers. The effect of the concentration of DMPA on Qs of MIP–NSHs was studied by varying the ratio in a range of 4:0 to 1:3 (v/v). The results showed that the best ratio of acetone to acetonitrile was 3:1, where Qs reached 2.24 μmol·g−1  can be prepared under the following conditions: the concentratration of hypericin: 5.0 mM; the concentration of monomer 1 and crosslinker 5 is 50 mM each; DMPA: 15.5 mM; solvent: acetone/acetonitrile in 3:1 (v/v), room temperature, UV light at 350 nm, 4 h. The MIP–NSH prepared under the optimal conditions described above, i.e. P14, were used for subsequent studies.Based on the results above, we conclude that the best combination of monomer/crosslinker for fabrication of MIP–NSHs in this work was The equilibrium adsorption isotherms of MIP–NSHs and NIP–NSs for the binding of hypericin were investigated by batch adsorption experiments. As observed in Q of the MIP–NSHs against that of NIP–NSs. Q of MIP–NSHs increased quickly in a linear relationship with the concentration of hypericin before Cs, a critical concentration of 11.5 μM. Thereafter, the increase of the Q slowed down and finally reached to a plateau. Furthermore, the amounts of substrate bound to the MIP–NSHs were more than that to the NIP–NSs, which was ascribed to the imprinting effect [The binding experiments were performed at different initial concentrations of hypericin, ranging from 0 to 50.0 μM, to compare the g effect .R2 is 0.9995, equilibrium constant Kd = 0.1843 μM, m is 1.8233, and Qmax is 6.07 μmol·g−1. The results indicate the absorption may be described by monolayer sorption on a non-smooth surface.Kd is the equilibrium constant (μM), Ce is the equilibrium concentration of hypericin (μM) in supernatant, and Qmax is the apparent maximum absorption capacity of binding sites (μmol·g−1).The adsorption mechanism study shows that the adsorption of MIP–NSHs towards hypericin fitted well with an extended Langmuir isotherm model  was obtained with MIP–NSHs compared to protohypericin and emodin . The binding selectivity of the NSs was evaluated with SF and IF, respectively. SF of MIP–NSHs towards protohypericin and emodin was 3.34 and 8.04, respectively; IF of MIP–NSHs towards hypericin, protohypericin, and emodin, was 2.44, 2.88, and 2.10, respectively  and fair selectivity towards hypericin. In addition, MIP–NSHs displayed good reusability up to at least five cycles. This work presents a straightforward method to fabricate molecularly imprinted nanospheres via thiol-yne click reaction. In conclusion, MIP–NSHs were successfully synthesized via thiol-yne click reaction using 3,5-diethynyl-pyridine as the monomer, tris(3-mercaptopropionate) as the crosslinker, and hypericin as the template. The click polymerization was completed in merely 4 h to produce the desired MIP–NSHs. The reaction conditions for adsorption capacity and selectivity towards hypericin were optimized, and the MIP–NSHs synthesized under the optimized conditions showed a high adsorption capacity (