KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma. The finex vivo. This NMDA model of RGC death has been utilized extensively to study earlier molecular beacons for the detection of RGC death in vivo and demonstrated the potential for the molecular imaging of RGC apoptosis in glaucoma [While the KcapTR488 sensor has been tested in cultured cells, detailed spectral analysis, caspase selectivity, and behavior in vivo have yet to be validated. Herein, the potential FRET properties of the reporter were explored in vitro and leveraged to characterize the caspase selectivity of the reporter with purified caspases. The NMDA model of caspase induction was utilized to test KcapTR488 in vivo using fluorescence imaging analysis glaucoma ,11,12,13The Institutional Animal Care and Use Committee at the University of Texas MD Anderson Cancer Center approved all animal experiments, which adhere to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in ophthalmic and vision research. Male Brown Norway rats weighing approximately 200 g each were purchased from Envigo.KcapTR488 was synthesized as previously reported ,10,12. SPrior to the biochemical analysis, the linearity and reproducibility of the in vitro assays were determined . Each coThe highly reproducible and linear response of the assay enabled the use of an overdetermined spectral unmixing algorithm to quantify the amount of product produced in vitro .To optically characterize the Texas Red and Alexa Fluor 488 intramolecular interactions in KcapTR488, 5 µM of KcapTR488 and Alexa Fluor 488 were dissolved in 1 X caspase3 assay buffer . Texas Red was initially dissolved in DMSO and then diluted to 5 µM in 1 X caspase 3 assay buffer. Caspase 3  was added to one set of KcapTR488 solution at a final concentration of 15 nM. Assays were conducted in triplicate in 96-well, clear-bottomed, clear-walled plates at a final volume of 100 µL. All samples were arranged in a pattern, leaving at least one empty well between adjacent samples. Assays were performed in a Synergy H4 microplate reader .For the Km and Kcat studies, caspase assays were performed at KcapTR488 concentrations ranging from 0.06 µM to 8 µM for caspase 1 (25 nM), 3 (0.14 nM), 7 (5.4 nM), and 8 (15 nM). Assays were performed at 37 °C. Blanks and zero-added enzyme controls were included for each experiment. The time series data were converted into the concentration of product generated per unit time via Intravitreal injections were performed as previously described ,13. Brie2 chamber. A transcardiac perfusion of 1000 units of heparin in 10 mL of 0.01 M PBS was followed by 4% paraformaldehyde in 10 mL of 0.01 M PBS. Whole eyes were harvested and fixed in 4% paraformaldehyde overnight at 4 °C. Following three 15 min washes in 0.01 M PBS, the retinas from the harvested eyes were dissected under a microscope. Whole-mount retinal tissue was prepared on slide glasses. For the preparation of retina cross sections, dissected retinas were further processed for cryoprotection. Retinas were washed in order with 5%, 10%, and 15% sucrose/0.1 M PBS solutions for 30 min and a 20% sucrose/0.1 M PBS solution overnight at room temperature. Retinas were placed into a 2:1 volume mix of 20% sucrose/OCT, respectively, for an hour and pure OCT for another 30 min. Retinas were then embedded in a cryomold with fresh OCT and slowly frozen on dry ice. Retinas were stored in a −80 °C deep freezer in zip lock bags until they were sectioned into 12 µm vertical cross sections.Rats were sacrificed in a COFor immunohistology staining, whole-mount retinas or retinal cross sections were permeabilized in 0.5% Triton X in 0.01 M PBS for 15 min at room temperature, then switched to a blocking solution of 0.1% Triton X and 1% BSA in 0.01 M PBS for 2 h at room temperature. For RGC marker staining, a primary anti-RNA-binding protein with a multiple splicing (anti-RBPMS) antibody , Cat#ABN1362) was diluted to 1:200 in blocking solution and added to the tissue for incubation at 4 °C for 4 days. After primary antibody incubation, tissues were washed three times for 15 min each in the blocking solution. The secondary antibody Alexa Fluor 647 , Cat#A-21245) at a 1:400 dilution in 0.01 M PBS was then added to the tissue and incubated at 4 °C overnight. RBPMS was found to be exclusively expressed in RGCs and displaced RGCs (dRGCs) and was therefore utilized as an RGC marker . For amaConfocal microscopic images were acquired under an Olympus FLUOVIEW FV1000 confocal laser scanning microscope using a PLAPO 60 X oil objective lens at a 12 µs/pixel sampling speed. For the detection of the sensor KcapTR488 signal, laser intensities were adjusted accordingly to minimize saturation and maximize positive signal pixels in both the FITC and Texas Red channels.The colocalization of fluorophores AF488 and Texas Red was accessed by ImageJ plugin JACoP . JACoP is a set of colocalization-calculating tools and includes Manders’s overlap coefficient, which we used to calculate the AF488 overlap with Texas Red under different conditions. The coefficient varies from 0 to 1, with 0 indicating nonoverlapping images and 1 reflecting 100% colocalization between images. When importing paired AF488 and Texas Red images to the plugin, thresholds of both images were calculated by adding three times the StdDev to the mean intensity of the selected background area. When selecting the background area, image edges were avoided. M1 and M2 values served as indicators of the colocalization of AF488 and Texas Red signals. M1 is defined as the ratio of the ‘summed intensities of pixels from the green image for which the intensity in the red channel is above zero’ to the ‘total intensity in the green channel’, and M2 is defined conversely for red. Therefore, M1 (or M2) is an indicator of the proportion of the green signal coincident with a signal in the red channel divided by its total intensity .In addition, we also calculated the fluorescence resonance energy transfer (FRET) ratio in the cell body to assess the proximity interactions between fluorophores AF488 and Texas Red within KcapTR488. For confocal microscopic scanning, excitation was set at 488 nm and emission was set at either 500–550 nm  or 595–645 nm . The FRET ratio equals the FRET signal divided by the FITC signal on a per-pixel basis. Cell body ROIs were then selected in ImageJ by creating a mask based on size and circularity. The average FRET ratio for each cell body, as defined in the mask, was quantified in the FRET image. The same process and values were utilized for all images.p = 0.0028, two-tailed Spearman test)  .Therefore, we confirmed that the spectral response at key excitation and emission pairs could be used as an indicator of KcapTR488 cleavage by recombinant caspases. RGCs, which are in close proximity to the vitreous in the ganglion cell layer (GCL), preferentially accumulate intravitreally delivered probes ,10,11,12To test the hypothesis that KcapTR488 can detect apoptosis in vivo, KcapTR488 was delivered via intravitreal injection into rat eyes undergoing NMDA-induced RGC death. In a healthy retina (no NMDA) from an animal sacrificed 2 h after KcapTR488 sensor injection A, there In a retina pretreated with NMDA and the animal sacrificed 6 h after reporter injection, yellow- and green-labeled cells in the GCL were noted C. HoweveFinally, The quantitative analyses of fluorophore colocalization and FRET under various conditions are summarized in In addition, FRET ratios were measured by the ratio of emission in the FITC spectral window over the Texas Red emission when excited by AF488 K. While Together, in healthy GCL neurons, these data demonstrated that the uptake of KcapTR488 was characterized by the nuclear localization of intact reporter. In the early stages of apoptosis, the cleaved NLS-Texas Red fragment subsequently exited the cell body in a subset of GCL neurons. In the late stages of apoptosis, the cleaved NLS-Texas Red fragment was not substantially transported down the axon and concentrated in the nucleus, while the AF488 fragment was evenly distributed in the cytosol. Therefore, KcapTR488 detected the progression of apoptotic cell death, as demonstrated by the changing subcellular distribution of fluorophores AF488 and Texas Red over time.Molecular beacons utilizing a fluorophore–quencher pair are optically silent in their intact state ,23. SuchUnlike molecular beacons that rely on a single channel for characterizing enzyme efficiency, the FRET signal from KcapTR488 enabled the rapid evaluation of caspase specificity in vitro . As expeIn addition to FRET readouts with KcapTR488, the sensor was amenable to quantitative analysis by dual-color localization. By this model, in healthy retinas in vivo, intact KcapTR488, like intact KcapQ488 and TcapQ488 ,25,26,27ex vivo fluorescence microscopy. After intravitreal injection, neurons in the GCL showed robust KcapTR488 uptake. The identity of these cells was confirmed by multicolor fluorescence imaging, as the cells were primarily RGCs but also included displaced amacrine cells. Note that while displaced amacrine cells in the GCL are common in rodents, in diurnal Anthropoidea, including humans, these cells are less prominent in the GCL [NMDA-mediated apoptosis was utilized to characterize KcapTR488 in vivo using  the GCL . As antip < 0.0001, d = 14). When compared to the FRET ratio, Manders’s overlap coefficient was more robust for detecting early changes in the apoptotic progression in the NMDA model of RGC death. Unlike analysis in cellulo, in which there is a closed homogenous compartment that contains all starting materials and products, a variety of transport and leak processes in vivo appear to render FRET less robust for analysis than changes in colocalization. For example, a loss of an AF488-cysteine product from the field of view or pixel that also retains intact reporter would result in an artificial net increase in the FRET efficiency and would therefore be interpreted as a “loss” of sensor activation and cell death, an incorrect conclusion both quantitatively and directionally. Additionally, the loss of both an AF488 product and an intact peptide with a rapid increase of TR fragment (>10 X) would also yield an artifactual increase in the FRET ratio. Other techniques such as hyperspectral fluorescence unmixing or 4–5 channel fluorescence unmixing might help disambiguate these cases by assigning an intensity and potentially concentration to each fragment. However, the same loss of AF488 fragment, or the loss of AF488 product and intact peptide, would not affect directionality of the overlap coefficient. Indeed, the loss of AF488-cysteine from the field of view would only appropriately enhance the change in Manders’s overlap coefficients, leading to the correct conclusion of apoptotic cell death.While qualitative analyses are important and informative, quantitative measurements are less subject to intra-observer and -operator variation. By design, changes in the subcellular localization of the NLS-Texas Red would be expected to reduce the correlation between the red and green channels. Furthermore, as demonstrated by the in vitro data, the cleavage of the DEVD sequence would be expected to yield changes in the pixelwise red/green ratio through decreased FRET. Indeed, the quantification of the colocalization could be achieved by Manders’s overlap coefficient analysis . The oveWhile leveraging the M1 and M2 overlap ratios at the whole-field level was superior to FRET at the whole-cell level, data suggest that this may not be the case if an appropriate FRET and multichannel scanning confocal ophthalmoscope were developed and commercialized for in vivo imaging at the mesoscopic scale. Such an instrument would allow researchers to take advantage of this reporter to exploit at least two different but biochemically related imaging metrics for an analysis arising from a single chemical entity. Due to the underlying nonhomogeneous and fractal nature of biological systems, all spatiotemporal data sets suffer from a special case of Simpson’s paradox ,29, wherAs consistently found with four previous generations of molecular beacon reporters, the change in subcellular compartmentalization also corresponded with TUNEL staining in a cross-sectional analysis . An analKcapTR488 has been extensively characterized in vitro and in vivo. The in vitro results demonstrated resonance energy transfer between AF488 and Texas Red, enabling a conventional ratiometric characterization of caspase cleavage efficiency and quantitative analysis through overdetermined spectral unmixing. The in vivo studies also confirmed the delivery of KcapTR488 to the GCL neurons, while quantitative changes in the colocalization of the fluorophores Texas Red and AF488 readily revealed the expected time-dependent activation of cell death following NMDA treatment. Unexpectedly, a quantitative analysis of changes in colocalization proved superior to a quantitative analysis of changes in the FRET ratio for differentiating NMDA-induced toxicity from healthy eyes as early as 2 h after the injection of the reporter.


The plants were sprayed with three JA levels, including 0, 5, and 10 mM JA. Salinity stress increased carotenoid and soluble protein content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. In contrast, salinity stress reduced plant height, leaf area, relative growth rate, proline content, and the activity of peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX). At the S2 salinity level, HA2 rate increased plant high by 9.7%, relative growth rate by 70.8% and CAT by 45.5, while HA1 increased leaf area by 12.5%, chlorophyll content by 22.3%, carotenoid content by 38.1%, SOD activity by 20.9%, MDA content by 18.0%, POD activity by 24.6% and APX value by 21.7%. At the S2 salinity level, the highest plant height, chlorophyll content, soluble protein content and APX value were recorded at 5 mM JA, while the highest leaf area, the content of carotenoid, proline, and MDA, and the activity of POD and CAT were achieved at 10 mM JA. Generally, 10 mM JA and 3 g HA kg−1 dry soil produced the best positive effects on forage sorghum plants physiological responses. Our study suggested that jasmonic acid and humic acid at appropriate rates can successfully mitigate the adverse effects of salinity stress on forage sorghum.Salinity is one of the primary abiotic stresses that cause negative physiological and biochemical changes due to the oxidative stress caused by the generation of reactive oxygen species (ROS). The effect of jasmonic acid (JA) as foliar spray and humic acid (HA) as soil amendment on the growth and biochemical attributes of forage sorghum plants exposed to salinity stress was investigated. Soil treated with NaCl at levels of 0, 2, and 4 g NaCl kg Antioxidioxygen . The actdioxygen . CAT is  oxidase .2O2 in Ricinus communis (L.) and a sharp formation of ROS at the initial moment of MeJA and ascribed it to the decrease in the enzymatic antioxidants activities. In another study with pomegranate (Punica granatum L.), Nicotiana tabacum L.) plants.Jasmonates (JAs), including methyl jasmonate (MeJA) and jasmonic acid (JA) can stimulate MDA accumulation and inhibit Fe-induced release of chelators that counteract salt stress. It is suggested that MeJA triggers some other protective mechanisms . JasmoniTriticum aestivum L.). Zea mays L.). Notably, humic acid can mitigate different stresses by increasing dry biomass weight and promoting plant growth (Humic acid (HA) is an organically charged bio-stimulant that significantly impacts plant growth and development . HA can t growth .Sorghum bicolor (L.) Moench) is one of the most important high-productive cereal crops . During the experiment, the average temperature was 31 °C, relative humidity was 76%, and cloud cover was 40%. Peat moss and vermiculite mixture (1:2 v/v) was used as the germinating and growing media of sorghum plants.−1 using a conductivity meter .The soil was collected from the surface of sandy loam soil (0–20 cm) of the Experimental Farm of Yangzhou University. The soil was presented as a sandy loam texture. The chemical and physical properties of the potting soil and chemical properties of HA are presented in th May in 2018, and on 15th May in 2019 in a seedbed for 15 days in the greenhouse. The strongest and uniform seedlings were selected and transplanted into pots. Each pot (30 cm in diameter × 32 cm in depth) was filled with 15 kg dry soil. On the 15th day after seedlings were transferred and applied with the NPK fertilizer (50 kg N/ha + 50 kg P2O5/ha + 50 kg K2O/ha). Another half dose was used on the 45th days of transplanting, and according to local recommendations, a spray of pesticides and weed control were conducted. Furthermore, the plants were watered regularly to maintain the water level every 3 days with a tap water.The seeds of forage sorghum variety Abu Sabeein were obtained from Agricultural Research Corporation, Khartoum, Sudan. The seeds were less than 8 months old and had been stored in paper bags under laboratory conditions (RH 40–60% at 15–20 °C). Seeds were surface-sterilized with 3% sodium hypochlorite solution for 1 min and then thoroughly rinsed three times with deionized water and air-dried near to their original weight for seeding. Seeds were germinated on 10All required approvals were obtained for the study, which complied with all relevant regulations.−1 dry soil (S2) . The subplots included three rates of humic acid, including 0, 3 and 6 g HA kg−1 dry soil, designed as HA0, HA1 and HA2, respectively. The sub–sub-plot included three levels of jasmonic acid, including 0, 5, and 10 mM JA.This study was a three-factorial experiment arranged in a split–split-plot randomized complete block design with three replications. The main plots included three salinity levels at 0 (S0), 2 (S1), and 4 g NaCl kgth day after seedling transplant, the plants at each NaCl level and humic acid rates were treated with exogenous jasmonic acid solutions as a foliar application. The spraying was repeated every 15th day. The plants were sprayed three times. During jasmonic acid application, care was taken to avoid any drift of different levels using a plastic shelter to separate each treatment. There were 27 treatments in the study with three replicates for each treatment. There were 81 pots, and all pots were placed in a greenhouse. Tap water (EC 0.26 dSm−1) was used. Pots were weighed every 2 or 3 days to maintain soil water content at 80% field capacity. The pots had no holes at the bottom to prevent drainage and leaching.Before seedling transplant, humic acid and NaCl at different levels were thoroughly mixed with the soil to make nine different treatments of salinity and HA. Before salinity and humic acid treatment, a 100 g soil sample was collected and oven-dried at 75 °C to constant weight, and the moisture content was calculated. On the 15th (Time one) and 70th (Time two) day after sowing. Three plants from every plot were randomly selected and tagged. Plant height was measured from a point immediately above the soil surface to the top of the plant. Four uppermost leaves including flag leaf from three plants of each pot were selected for measuring leaf area with a leaf area meter . The relative growth rate was calculated according to Plant height (cm) was recorded on the 50th day of seedling transplant, a healthy leaf from the middle of the plant excluding the four uppermost leaves, was harvested and immersed in liquid nitrogen for 20 min and stored in a low-temperature freezer to determine the activity of enzymes and the content of proline and soluble protein. Leaf protein was extracted using a phosphate buffer solution containing sodium phosphate dibasic dehydrate and sodium phosphate monobasic dehydrate. Stored leaf tissue (0.2 g) was crushed in 2 ml of the phosphate buffer solution, and the slurry was centrifuged at 10,000g for 20 min at 4 °C. The supernatant was used for the determination of the activities of enzymes including SOD, CAT, POD, and APX.On the 50The activity of SOD and CAT was determined following the method of Soluble protein was estimated for each extract . A dye sProline content was estimated using the protocol of The determination of photosynthetic pigments such as total chlorophyll content and carotenoid content were conducted according to the method reported by F values were significant, means were separated by the least significant difference (LSD) test (P ≤ 0.05) as described by This study was performed in two different seasons, and there were no significant differences in all the parameters between the two seasons . TherefoThe results revealed that jasmonic acid, humic acid, salinity, and their interactions significantly affected most parameters measured on most occasions  and 3.The growth parameters such as plant height, leaf areas and relative growth rate were significantly decreased with increased salinity rate. In the interaction between salinity and humic acid, at the control of humic acid (HA0), the high salinity rate of S2 reduced the plant height by 9.1% , leaf ar2 , while S0  showed the lowest value (1.65 mg g−1 FW) , the high soil salinity rate of S2 decreased the chlorophyll content by 43.4%  in compa g−1 FW) . The chl g−1 FW) , and car g−1 FW) , in compSoluble protein and proline content were significantly decreased with increased salinity rates. In the interaction between salinity and jasmonic acid, at the control of jasmonic acid (0 mM JA), the high salinity rate of S2 reduced the proline content by 54.6% , in comp−1 FW) and carotenoid content (1.77 mg g−1 FW) (The growth parameters (plant height and relative growth rate) and photosynthetic pigments (chlorophyll content and carotenoid) were affected by the interaction between jasmonic acid and humic acid . Accordi g−1 FW) .−1 min−1 , high salinity rate of S2 decreased POD activity by 33.8% , CAT by −1 min−1  and MDA −1 min−1 . The act−1 min−1 , MDA con−1 min−1 , POD act−1 min−1 , and APX−1 min−1 , in comp−1 min−1  over the−1 min−1  and CAT activity (40.6 U g−1 min−1). However, sorghum plants treated with HA2 + 10 mM JA recorded the highest SOD activity (25. 7 U g−1 min−1) and POD activity by 66.7 U g−1 min−1. On the other hand, the treatment of JA0 + HA0 recorded the lowest content of soluble protein and the lowest activities of most antioxidants enzymes , In the current study, NaCl salinity stress significantly inhibited plant growth traits of forage sorghum, including plant height, leaf area, and relative growth rate. The inhibition in the growth parameters may be due to osmotic impacts of salt stress or increments in growth retardants, water imbalance , ions toIn this study, leaf area was decreased with increasing salinity stress. The decrease in leaf area under salinity stress has been attributed to suppressed cell division, the shrinkage of the cell contents leading to reduced development and differentiation of tissues, unbalanced nutrition, and disturbed avoidance mechanism . In thisRelative growth rate depends on canopy photosynthesis per area of land. In the present study, the relative growth rate of salt-stressed plants in the high salinity treatment was lower than that of the other salinity treatments. Decreases in plant growth under salinity stress might be endorsed to the reduction in water absorption due to reduced osmotic pressure in the soil solution . Our resPlant hormones play essential roles in stress responses and adaptation. It is clearly defined that jasmonic acid (JA) increased in response to salinity . In thisOrganic fertilizer application such as vermicompost and humic acid were optimal for improving soil properties because of the increased organic matter, nutrient contents, the vital role of soil enzyme action, improved soil aeration, and enhanced microbial action in soil, thus improving soil physical and chemical properties . Humic aThe chlorophyll content is widely used as an index to indicate the abiotic tolerance level in plants. Protection of chloroplast and photosynthetic machinery is the first target of defense under stressful conditions . In thisIn the present study, jasmonic acid application increased the total chlorophyll content and carotenoid content under saline conditions, which is one of the factors contributing to higher photosynthetic of forage sorghum plants under salinity conditions. Our results were similar to those of The stimulation in photosynthetic pigments caused by humic acid caused may be due to the decrease of pH value and increase in the activity of soil organisms which release more nutrients from the soil such as Fe . In the Improving soluble protein and proline content are an important mechanism that plants use to alleviate the harmful effect of salinity stress . In the In the study, humic acid improved the contents of soluble protein and proline of forage sorghum. Our results were dissimilar with Jasmonic acid can protect the plant from toxicity ions in the different stages by managing the antioxidant machinery and synthesis of proteins . In the 2− and is considered an essential antioxidant in cells. In our study, high salinity stress increased SOD activity and MDA content. The increased in SOD activity coincided with an increasing in the activities of Mn-SOD and Fe-SOD (Gypsophila oblanceolate plant. Similar results were noticed by Superoxide dismutase (SOD) is one of the enzymes responsible for eliminating Od Fe-SOD . These rOur study showed that significant increases in SOD activity and MDA content were observed in forage sorghum plants treated with JA. The increase in SOD activity agreed with the results of In our results, the application of HA under saline condition increased SOD activity and MDA content. Different result was showed by 2O2 accumulation, which could result in a Haber–Weiss reaction from hydroxyl radicals , and hot pepper  plants under salt stress. Also, Under salt stress, the APX plays an essential role in protecting plants by reducing H2O2 . Our stu2O2 . Kesba &−1 soil decreased all the parameters tested, except for protein content, SOD activity, and MDA content. Jasmonic acid significantly improved salt stress tolerance in forage sorghum plants; and the plants sprayed with 10 mM JA level had higher POD and SOD activities. Among different humic acid rates, 3 g HA kg−1 dry soil successfully increased all the parameters tested. Generally, 10 mM JA and 3 g HA kg−1 dry soil produced the best positive effects on forage sorghum plants physiological responses. Our study suggested that proper management of humic acid as soil amendment and jasmonic acid as foliar spray could be conducted in salt-affected soils to sustain forage growth and increase crop yield and productivity of forage sorghum.Our study examined the effects of different rates of humic acid as soil amendment and jasmonic acid as foliar spray on growth parameters, total chlorophyll content, carotenoid content and antioxidant enzyme activities of forage sorghum exposed to salinity. High soil salinity rate of 4 g NaCl kg10.7717/peerj.13793/supp-1Supplemental Information 1Click here for additional data file.


After 60 days, fish were subjected to crowding stress (80 kg/m3) for 24 h. The growth of fish was highest in MD35. The feed conversion ratio was lower in MD35 compared to the control and HD groups. The amylase, lipase, protease, and superoxide dismutase and glutathione peroxidase activities in the biofloc groups were significantly higher than in the control. After crowding stress, cortisol and glucose levels in biofloc treatments were significantly decreased compared to the control. After 12 and 24 h stress, lysozyme activity in MD35 was significantly lower than in the HD treatments. Overall, the biofloc system with MD could improve growth and robustness against acute stress in fish. Also, biofloc could compensate 10% reduction of protein in common carp juvenile diet when reared in MD.This study is aimed at evaluating the effects of stocking densities and dietary protein levels in the biofloc system on the performance of common carp. Fish (12.09 ± 0.99 g) were transferred to 15 tanks: fish reared at 10 kg/m Also, tous food , 5. The ous food , 7. Biofous food , 5.3 for the intensive cultivation of this species in biofloc conditions. Optimal density plays an important role in raising profit of fish production; thus, investigation is needed to determine the most suitable rearing density for better performance in the biofloc conditions. Moreover, it was reported that the crowded  common carp was more susceptible to an additional acute confinement stress [Fish farmers try to raise productivity and profitability by increasing the fish stocking density, despite its unfavorable effects on farmed fish , 9. In tt stress . Acute st stress . So, furOreochromis niloticus) fed 20% dietary protein and cultured in the biofloc system with wheat milling by-product significantly surpassed those fed 30% dietary protein and cultured in a clear water system [Rhynchocypris lagowski. So, preparing low-protein diet decreases production costs and the influence on the environment by decreasing nitrogen input and fish meal usage in the diet  fingerlings were obtained from a local fish farm . The fish were kept in two fiberglass tanks  and fed a diet containing 35% crude protein  and 20 kg/m3 as high stocking density . The trial was designed in a 2 × 2 factorial design with two stocking densities (10 and 20 kg/m3) and two dietary crude protein levels (35 and 25%) in a biofloc system. Overall, four biofloc and one clear water (control) treatments (with three replicates of each one) were established: fish reared at medium density and fed diets containing 35% (MD35) or 25% (MD25) crude protein and fish reared at high density and fed diets containing 35% (HD35) or 25% (HD25) crude protein in the biofloc system and control fish (reared at medium density and fed a diet containing 35% crude protein in clear water system).The experiment was performed at the fisheries laboratory, Faculty of Agriculture and Natural Resources, Gonbad Kavous University , in compliance with the ethics and University guidelines for the care and use of animals. Common carp  tanks were initiated as described by Adineh et al. . BrieflyFish were hand-fed the experimental diets three times a day  for 60 days at 2.5% of the total fish biomass in each tank, regulating the daily amount of food every two weeks based on the fish biomass. In the clear water (control) group, no carbon source was added to the tanks, and approximately 40% of the water was replaced every two days. In the biofloc tanks, freshwater was only added to compensate water loss caused by evaporation. Moreover, the biofloc tanks were supplied daily with carbon source (beet molasses) to adjust the C : N ratio of 15 : 1 for optimum production of biofloc according to Crab et al. [Two diets with 35 and 25% crude protein levels were formulated for common carp fingerlings . All ingAt the end of the trial, the biofloc produced in biofloc tanks were collected, settled, concentrated, dried in an oven at 105°C, and then subjected to a chemical composition analysis. The proximate composition of the experimental diets and biofloc samples was analyzed in triplicates according to the standard methods .Water temperature, salinity, dissolved oxygen, electrical conductivity (EC), and pH were measured daily in the tanks using a portable multimeter . Total dissolved solids (TDS) were analyzed daily . Total ammonia nitrogen (TAN), nitrite, and nitrate levels were calorimetrically determined every 10 days following standard methods . Total sWeights of all fish from each tank were determined at the end of the 60-day trial after 24 h of starvation. Then, growth and feed utilization parameters were calculated. Also, 4 fish from each tank (12 fish per treatment) were randomly captured and anaesthetized with 200 mg/L clove powder [3) for 24 h by decreasing the water level in tanks in a similar method to Adineh et al. [After 60 days of rearing and sampling, all fish from control and biofloc tanks were subjected to crowding stress . Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities were measured using assay kits .The levels of serum cortisol were measured by the competitive enzyme-linked immunosorbent assay (ELISA) procedure using a commercial test kit . Serum glucose content was determined based on the glucose oxidase procedure using a test kit . The level of serum lactate was also determined using a commercial test kit  according to the manufacturer's instructions. Serum lysozyme activity was measured by the turbidimetric analysis , based op < 0.05 was the agreed significance level. All the results were presented as means ± standard deviation(SD). Before analysis, the Shapiro-Wilk and Levene's tests were performed to confirm the normality and homogeneity of variance of the data, respectively. Then, the data were analyzed by one-way analysis of variance (ANOVA) to determine the significant differences between groups, followed by Duncan's multiple range tests to compare the means between groups. Two-way ANOVA was applied to check the effects of stocking density, dietary protein level, and their interactions excluding the clear water (control) group. Significant differences between the means achieved before and after crowding stress were analyzed by one-way ANOVA followed by Dunnett's test [t-tests treat one group as a control (before stress) and compare other groups  against it. The following parameters were also calculated:Statistical analysis was done using SPSS software version 24.0, and Water quality parameters in biofloc and control tanks during the 60-day trial are shown in On day 50, MD35 and HD35 biofloc treatments had significantly higher TAN values than the control (clear water) group . On day The proximate composition of biofloc in MD and HD biofloc tanks stocked with common carp and fed 35 and 25% dietary protein for 60 days is shown in Growth and feed utilization parameters of fish stocked in biofloc systems and control (clear water) for 60 days are presented in Digestive enzyme activities in the digestive tract and antioxidant enzyme activities in the liver of fish cultured in biofloc systems and control (clear water) for 60 days are shown in The liver SOD and GPx activities in fish cultured in all biofloc groups were significantly higher than in the control (clear water). The liver catalase activity in MD35, MD25, and HD35 groups was significantly higher than in the control and HD25 groups. The liver TAOC and MDA levels were significantly affected by stocking density, dietary protein level, and the interaction of these two factors. The liver TAOC level was highest in the MD biofloc groups and lowest in the HD25 group. The liver MDA content was significantly higher in the control and HD25 groups, then the HD35 group, and lower in the MD biofloc groups.3) for 24 h are presented in Cortisol, glucose, and lactate levels and lysozyme, ALT, and AST activities of fish cultured in medium and high densities and fed 35 and 25% dietary protein in a biofloc system and control  for 60 days and after acute crowding stress . On day 60, the lowest TAN levels were observed in the control and MD biofloc groups. Previous studies also showed that TAN concentration was effectively reduced in the BFT groups [−1) and control groups on day 50. On day 60, nitrite concentration was significantly lower in the control and MD25 groups compared to the HD biofloc groups. On day 60, higher stocking density led to higher TAN and nitrite values because of the higher usage of food and more fish excretion. High stocking density combined with the high dietary protein content in HD35 tanks negatively affects the water quality particularly the accumulation of ammonia and nitrite. Although the TAN and nitrite tended to raise in the biofloc groups, the formation and growth of the biofloc in the BFT system were associated with the assimilation of nitrogen substances by heterotrophic bacteria or autotrophic nitrification [−1 [−1 in culture water might cause death to the farmed fish. However, nitrite quickly converts to the more stable form (nitrate) in biofloc environment. Biofloc groups had significantly higher nitrate values than the control (clear water), because the nitrifiers were active in the biofloc tanks ([A meaningful decline in pH related to the high abundance of bacteria (higher TSS and BVF levels) in HD biofloc tanks was showed. Bacterial respiration apparently raised the COne in pH , 33. Totne in pH , 33. In ne in pH . TAN, niT groups , 35. Morfication , and thetion [−1 , 19. Boytion [−1  also repc tanks . Therefin situ biofloc utilization by fish might have an impact on the proximate composition of biofloc . In both MD and HD biofloc groups, higher lipase activity was observed in 35% dietary protein groups. Similarly, Khorasaninasab, Keyvanshokooh, Pasha-Zanoosi, and Shahriari  exhibite3 stocking densities were significantly lower than the fish cultured in the clear water tanks at 12 kg/m3 stocking density. Also, elevated SOD and catalase activities were reported in Nile tilapia cultured in biofloc conditions compared with fish cultured in clear water [in situ had anticrowding stress efficacy [The stress response may raise free radicals, resulting in higher lipid peroxidation levels and further oxidative damage . Antioxiar water . In the efficacy , 9. In aefficacy .R. lagowski. Moreover, lower levels of cortisol and glucose and higher lysozyme activity were reported in biofloc tanks at stocking densities up to 250 fish/m3 compared to the control  [Stress that induces elevated blood cortisol levels can also suppress immune responses of fish . Before fish/m3) . Higher fish/m3) . Accordifish/m3) , lysozymfish/m3) , 47, 48.fish/m3) , 49. Thein situ had anticrowding stress efficacy. We concluded that common carp reared in biofloc conditions could raise the ability to resist stress. Moreover, our findings exhibited that in the HD biofloc groups, 35% dietary protein (HD35) is more efficient than 25% dietary protein (HD25) in preventing the increase in cortisol levels induced by crowding stress.After 12 and 24 h of crowding stress, serum cortisol and glucose levels in all treatments were significantly higher than the prestress values. At all time points after acute stress, cortisol and glucose values in the biofloc treatments were significantly decreased compared with the control (clear water). These results indicated that biofloc could alleviate the elevation in cortisol and glucose caused by acute stress. This was confirmed by the results of Fauji et al. , showingIn the present study, increased stocking density influenced the acute crowding stress response in common carp. After 24 h crowding stress, cortisol and glucose values in HD biofloc groups were significantly higher than in the MD biofloc groups, and serum lactate levels in the HD biofloc groups were significantly higher compared to the MD35 biofloc group. These results reveal that common carp reared in HD biofloc tanks were more susceptible to acute crowding stress compared to the MD biofloc groups. In agreement with our findings, after 15 h crowding, plasma cortisol concentrations of common carp in crowded plus confinement treatments were higher than in the control and confinement treatments . Serum lAfter 12 and 24 h stress, lysozyme activity in HD treatments was significantly higher than before stress. The fish in HD treatments may have enhanced lysozyme activities to further stress resistance . SimilarThe activities of ALT and AST in serum can be used as important indicators of the function of the fish liver, as the increased permeability of damaged hepatocytes causes a release of these enzymes into the blood . These e3 could be recommended. Also, microbial flocs as a protein source could compensate 10% reduction of protein in common carp juvenile diet when reared in MD.In conclusion, our study demonstrated that the growth of juvenile common carp in the HD biofloc groups was significantly lower than in the control, while FCR was increased in the HD25 biofloc group compared to the control, suggesting an inefficiency of biofloc for fish growth in HD. Our results revealed that the biofloc system with medium stocking density could reduce oxidative stress and improve growth performance, feed utilization, digestive activity, immune status, health, and resistance to additional acute stress in fish. Furthermore, high dietary protein levels could improve growth, lipase activity, antioxidant status, and stress response. Therefore, the culture of juvenile common carp in the biofloc system at an initial stocking density of 10 kg/m


Our study demonstrated that USPIO labeling was a feasible approach for in vitro tracking of brain organoid-derived EVs, which paves the way for further in vivo examination.The significant roles of extracellular vesicles (EVs) as intracellular mediators, disease biomarkers, and therapeutic agents, make them a scientific hotspot. In particular, EVs secreted by human stem cells show significance in treating neurological disorders, such as Alzheimer’s disease and ischemic stroke. However, the clinical applications of EVs are limited due to their poor targeting capabilities and low therapeutic efficacies after intravenous administration. Superparamagnetic iron oxide (SPIO) nanoparticles are biocompatible and have been shown to improve the targeting ability of EVs. In particular, ultrasmall SPIO  are more suitable for labeling nanoscale EVs due to their small size. In this study, induced forebrain neural progenitor cortical organoids (iNPCo) were differentiated from human induced pluripotent stem cells (iPSCs), and the iNPCo expressed FOXG1, Nkx2.1, α-catenin, as well as β-tubulin III. EVs were isolated from iNPCo media, then loaded with USPIOs by sonication. Size and concentration of EV particles were measured by nanoparticle tracking analysis, and no significant changes were observed in size distribution before and after sonication, but the concentration decreased after labeling. miR-21 and miR-133b decreased after sonication. Magnetic resonance imaging (MRI) demonstrated contrast visualized for the USPIO labeled EVs embedded in agarose gel phantoms. Upon calculation, USPIO labeled EVs exhibited considerably shorter relaxation times, quantified as T Human induced pluripotent stem cells (iPSCs) have been a desirable replacement for embryonic stem cells since Yamanaka’s breakthrough . iPSCs hUpadhya et al.  derived Due to their significant roles not only as therapeutic agents ,22,23 buIn the engineering of EV cargo, it is critical not to disrupt the integrity of the EV membranes during the labeling in order to preserve their physiological functions ,34. SPIOIn our previous work , NPC org2 and T2* contrast. This work could serve as a foundation for future injection of forebrain organoid-EVs in animal models such as ischemic stroke, which can be tracked by MRI.In this paper, iNPCo was derived from iPSCs, and characterized for several key markers of the organoids. Then, the isolated EVs from the iNPCo condition media were loaded with USPIOs in EVs by sonication for 30 s in total, followed by incubation at 37 °C for one hour. EVs with or without USPIOs were characterized in terms of size, concentration, morphology, and miRNA expression. Besides, in vitro MRI analysis was performed for the USPIO-labeled EVs verses unlabeled EVs, showing the T5 cells/well in differentiation medium composed of DMEM/F-12 plus 2% B27 serum-free supplement . iPSK3 cells were seeded in the presence of Y27632 (10 μM). After 24 h, Y27632 was removed and the formed embryoid bodies (EB) were treated with dual SMAD signaling inhibitors of 10 μM SB431542  and 100 nM LDN193189 (Sigma) over 7 days. Then, on day 8, the spheroids were treated with fibroblast growth factor (FGF)-2  and cyclopamine  for cortical differentiation for up to 21 days . To assess the statistical significance, one-way ANOVA or student’s The differentiation timeline of the forebrain organoids (iNPCo) was described in The organoids were replated for 8 days, and the conditioned media were collected every two days. The process of EV isolation was shown in TEM imaging showed the double−layer cup−shape morphology of the EVs A. The da2 and T2* values were extracted from the average signal intensity and time profiles for each sample by fitting the respective data to a first exponential decay. As seen in 2 and T2* relaxation were established between sample layers, likely as a result of partial volume effects. This preparation method was repeated with similar outcomes using 8–10 nm of SPIO, as can be seen in The feasibility of utilizing MRI to visualize and compare SPIO−labeled EVs was assessed. Agarose gel phantoms are commonly used to induce longitudinal and transversal relaxation rates comparable to that of in vivo tissues ,51. In v2 approximately 26.3% and T2* 43.5% compared to unlabeled EV. Interestingly, unlabeled EV lengthened T2 and T2* values by 15.5 and 30.6%, respectively, compared to gel only. Similarly, T1 saturation was also assessed, and values were extracted from the average signal vs. recovery time profile by fitting an exponential rise to maximum. As expected, T1 effects remained minimal with labeled EVs decreasing T1 only 6.4%. To minimize partial volume effects, the gel volume of EV layers was reduced, increasing the EV concentration within each layer. As demonstrated in 2, T2* and T1 values, as expected, this preparation resulted in 63.6, 68.1 and 19.5% reductions, respectively, for the labeled EVs. It is important to note that the region of interest used in the analysis avoided the central region of inhomogeneity caused by gel artifact. A more homogenous layer of SPIO−generated contrast within the labeled EV layer was prepared by exposure to the SPIO of 5 nm at 0.5 mg/mL followed by ExtraPEG removal of free SPIO . SimilarThis study derived iNPCo from iPSCs using a dual SMAD inhibition method . The orgHaving derived iNPCo, the secreted EVs were isolated from the conditioned medium of the replated culture using the inexpensive ExtraPEG method . FollowiThe EV morphology and the internal USPIO were examined by TEM. The particles were found to be still double layered, with black dots inside, revealing the successful involvement of USPIO in EVs. The specific miRNA cargo after USPIO labeling was examined by RT-PCR. Four out of six tested miRNAs had decreased abundance after the labeling. Further optimizations of the labeling process to minimize the cargo loss during USPIO labeling are necessary. In many studies, USPIO were loaded into EVs through incubation with cells ,59,60,612 effect, ultimately reducing signal and giving rise to the dark regions seen in the MR images provided in 2*−weighted MRI is used to optimally visualize this contrast. Here, the effects of iron oxide size and EV preparation were investigated using MRI of the USPIO−labeled organoid−EVs embedded in tissue−mimicking gels. In the first preparation method, which consisted of 15–20 nm USPIO labeled EVs, partial volume effects contributed to low contrast within the labeled EV gel layers. This was corrected for in subsequent samples by decreasing the volume of gel within the EV layers. As a result, a more uniform layer of labeled EVs with higher MR contrast was generated, albeit in a punctate distribution suggesting potential aggregation of the EVs. In order to quantify the contrast generated by the USPIO, T2 and T2* values were extracted from their respective exponential signal decays. Although more easily visualized compared to the previous preparation method, T2 and T2* effects remained limited. Further optimization was pursued by reducing the USPIO size to 5 nm and incorporating the previously defined ExtraPEG method to remove free USPIO prior to embedding in the gel. This shortened the T2 and T2* values resulting in significantly increased MRI contrast. Although the varying concentrations of labeled EVs require discretion in comparing between the first and second agarose gel preparations, the enhanced MRI contrast observed with the smaller USPIO size demonstrates the feasibility of labeling forebrain organoid−EVs using USPIO for non−invasive MRI toward potential animal study, such as ischemic stroke rat models. Visualization and tracking of EVs would be beneficial to understand their bio distribution potential and MRI is particularly suitable to this goal as it has already been employed successfully to track stem cells labeled with larger iron oxide particles in various disease models including ischemic stroke ,70. USPI2 and T2* values compared to unlabeled EVs and gel only. Our study demonstrated that USPIO labeling was a practical strategy for in vitro tracking of EVs, which paves the way for further in vivo examination.In this study, we generated forebrain organoid iNPCo from iPSC, and isolated EVs from the conditioned medium of the culture. The EVs were labeled with USPIO by the sonication method. Their size distribution did not change significantly, but their recovery and some of the miRNA cargo decreased after sonication. Though feasible, this labeling approach should be modified to reduce the loss of EV particles and EV cargo. MRI confirmed contrast visualization only for the USPIO labeled EVs embedded in agarose gel phantoms. USPIO labeled EVs exhibited shorter T


Hyperspectral technology has enabled rapid and efficient nitrogen monitoring in crops. However, most approaches involve direct monitoring of nitrogen content or physiological and biochemical indicators directly related to nitrogen, which cannot reflect the overall plant nutritional status. Two important photosynthetic traits, the fraction of absorbed photosynthetically active radiation (FAPAR) and the net photosynthetic rate (Pn), were previously shown to respond positively to nitrogen changes. Here, Pn and FAPAR were used for correlation analysis with hyperspectral data to establish a relationship between nitrogen status and hyperspectral characteristics through photosynthetic traits. Using principal component and band autocorrelation analyses of the original spectral reflectance, two band positions (350–450 and 600–750 nm) sensitive to nitrogen changes were obtained. The performances of four machine learning algorithm models based on six forms of hyperspectral transformations showed that the light gradient boosting machine (LightGBM) model based on the hyperspectral first derivative could better invert the Pn of function–leaves in cotton, and the random forest (RF) model based on hyperspectral first derivative could better invert the FAPAR of the cotton canopy. These results provide advanced metrics for non–destructive tracking of cotton nitrogen status, which can be used to diagnose nitrogen nutrition and cotton growth status in large farms. Niton crops . In addion crops ,14. The on crops ,19,20,21on crops  or opticon crops ,24. SuchRemote sensing monitoring, as the current mainstream mode of detection, has the unique advantages of being rapid, non–destructive, and multidimensional, and is widely used in the agricultural production process ,26,27,28The aim of this study was to construct a remote sensing estimation model for photosynthetic characteristic parameters and population photosynthetically active radiation in cotton leaves under different nitrogen levels. During the key growth period of cotton, the photosynthetic parameters of cotton leaves, population photosynthetically active radiation, and canopy spectral information were obtained. By mining spectral information, the optimal modeling method and hyperspectral variables were used to construct an estimation model of the photosynthetic characteristic parameters of cotton leaves and the population’s photosynthetically active radiation. This study provides a theoretical basis for efficient and non–destructive monitoring of the nitrogen nutrition status of cotton and provides scientific guidance for specifying scientific and reasonable fertilization programs.2·m−2·s−1 and 0.7949–0.9925, respectively , root mean square error (RMSE), and mean absolute percent error (MAPE), which are indicators of model performance , random forest (RF), extreme gradient boosting (XGBoost), and light gradient boosting machine (LightGBM). Model performance was evaluated using the ratio of performance to deviation (RPD) values and robustness to multiple repetitions. The machine learning models indicated high performance and robustness when first–derivative reflectivity and second–derivative reflectivity were applied as hyperspectral data in the training and validation datasets. For Pn modeling, RF, XGBoost, and LightGBM models indicated high performance and robustness when FD was applied as hyperspectral data both in the training data and validation data, as most RPD values were greater than 1.4, representing an acceptable prediction level A. For FAformance .Nitrogen management is problematic in large–scale cotton cultivation. However, laboratory methods for measuring nitrogen content and photosynthetic characteristics remain complex experimental techniques that are not suitable for large–scale and real–time monitoring ,23,24. THyperspectral techniques can obtain detailed spectral information from plants and can be used to closely monitor plant growth as well as physiological and biochemical properties ,50,53,56At the same time, it is necessary to use preprocessing methods to improve the level of hyperspectral modeling. Preprocessing of original spectral data was performed using the first derivative, second derivative, wavelet transform smoothing, Savitzky–Golay smoothing, and natural logarithm transformation to reduce the effect of background noise. These preprocessing methods, based on the full–band original spectral dataset, retained all the information of the original spectral data. Our research shows that the accuracy and stability of the prediction model were improved by using suitable preprocessing and transformation and that the machine learning model built using derivative–type spectral data was better than other preprocessing methods. Peng et al. believed that when the distance between the sensor and the collected crops was small, the low–order differential could effectively remove spectral background noise and improve the inversion ability of the model . Xia et Based on a powerful and flexible algorithmic framework, machine learning has the potential to analyze hyperspectral reflectance data, thereby avoiding the waste of hyperspectral information in traditional research ,65,66. IThe machine learning methods LightGBM, RF, and XGBoost showed high performance and robustness for both Pn and FAPAR. The excellent performance of LightGBM may be attributed to its unique leaf growth strategy. RF is more robust for input variables and outliers. XGBoost adds a regular term to control the complexity of the model and reduce the possibility of overfitting. Therefore, machine learning is a powerful tool for estimating crop–related indicators using hyperspectral technology. In recent years, machine learning models have been increasingly used in agricultural remote sensing ,65,66,67The experiments were conducted from May to September 2019 (Test2019) and 2021 (Test2021), respectively, in two experimental fields with different basal fertilities  at the f−1; (iii) common nitrogen application (N2), N fertilizer applied at 300 kg N ha−1; and (iv) excessive nitrogen application (N3), nitrogen fertilizer applied at 450 kg N ha−1. The sub–area treatments with cotton of two different genotypes were as follows: (i) Xinluzao61 and (ii) Xinluzao72.The experiment was conducted using a split–plot experimental design of randomized groups with three replicates, which included treatments with four amounts of nitrogen fertilizer as follows: (i) No nitrogen application (N0), without N fertilization application; (ii) less nitrogen application (N1), N fertilizer applied at 150 kg N ha2O5 ha−1 and 75 kg K2O ha−1. The application rates were recommended by local agricultural technicians and were sufficient to meet the requirements of cotton. The fertilizer types used in this study were urea (46.4% N), potassium dihydrogen phosphate , and potassium sulfate (52% K2O). All fertilizers were applied in multiple drops of water  and potassium (K), were the same for all treatments: 105 kg Pof water .−1. Weeds, pests, and diseases were controlled using chemicals via local methods. No obvious weed, pest, or disease problems occurred during the cotton–growing season.Two cotton varieties were selected (Xinluzao 61 and Xinluzao 72); both have excellent variety characteristics and are the major cultivars in Xinjiang . A plast2·m−2s−1) of cotton key functional leaves was measured on clear and cloudless days during five reproductive periods using an LI–6400XT portable photosynthesis instrument . The data were collected on the 55th, 70th, 85th, 100th, and 115th days after cotton emergence, involving the full bud stage, full flowering stage, flower and boll stage, full boll stage, and early boll–opening stage of cotton. The effective radiation of the light source was PAR 1800 μmol·m−2s−1, the leaf chamber used was 2 × 3 cm, the gas flow rate was 500 mmol·s−1, and the CO2 injector was 400 μmol·mol−1. Ten plants from each treatment group were centrally selected for each measurement period.Following the standard method , cotton PAR was synchronously sampled with the leaf net photosynthetic rate and measured in the time domain. PAR was measured using an LI–250A light meter with an LI–190SA quantum sensor . According to previous methods ,24, the For synchronization with the measurement of photosynthesis, the spectral radiance of the cotton canopy was measured with a full–range hyperspectral SR–3500 portable ground object spectrometer  under natural light on a sunny day in the field. The instrument collected data in the 350–2500 nm spectral range, with a resampled spectral resolution of 3 nm before 1000 nm, 8 nm between 1000 and 1500 nm, and 6 nm after 1500 nm.For cotton plant canopies where photosynthesis was measured, the spectral reflectance was measured 50 cm above the cotton canopy using a hyperspectral one–handed remote–measuring device. The acquired reflectance spectra could be affected by several environmental factors. Whiteboard calibration was used to eliminate or minimize these side effects . Before Owing to background noise, the signal–to–noise ratio of the original spectral reflectance was low. Smoothing, derivative processing, and mathematical transformation are considered to be important for improving the signal–to–noise ratio ,72. To bUsing the splice correction function that comes with the instrument, reflectance data at 1–nm steps were obtained across the entire wavelength domain from 400 to 2500 nm. Hyperspectral data contain information on many bands and there is large data redundancy and collinearity, which can affect model building ,58. ThusPrincipal component analysis can analyze the main factors that cause differences in a large number of variables, and then achieve the effect of data dimensionality reduction ,74. By s2 correlation coefficient was calculated using a pairwise combination, and all the combinations constituted a 2151 × 2151 r2 matrix. By selecting the band combinations corresponding to 2000 smaller values in the r2 matrix of different growth periods, the occurrences of different bands were counted, and the band range with rich hyperspectral information was determined according to the band distribution.Band autocorrelation can reduce spectral content redundancy through the correlation relationship between the two bands . The smaHowever, various methods have been devised to select the range of characteristic bands, each with its own advantages and limitations. Therefore, in this study, we consider the common part of the characteristic bands screened by the two methods as the characteristic band range.The process of generating regression models is described in a traditional manner , with slBased on the feature bands screened by band autocorrelation analysis and principal component analysis, correlation estimation models of cotton leaf Pn and canopy FAPAR were constructed using four different machine learning algorithms, including SVM, RF, XGBoost, and LightGBM with spectral OR, FD, SD, WT, SG, and NL as independent variables.SVM can better minimize the structural risk and select the optimal solution between the accuracy of the given data and the complexity of the function to obtain the best model promotion ability. The SVM model has the advantage of solving the problems of the small sample size and nonlinear, high–dimensional data .The decision tree is a common machine learning algorithm that is easy to understand and interpret. RF builds a bagging ensemble with a decision tree as the base learner and further introduces random feature selection in the training process of the decision tree, which has great advantages in processing high–dimensional data . XGBoostThe preprocessing of the five types of hyperspectral data was performed using Origin 2022b  and Microsoft Excel 2019 . The SG smoothing window size was set to 5 . We set Four machine learning algorithms were performed using the “mlr3” package (V. 0.13.3) in R 4.1.3 . This pa2, RMSE, and MAPE, which were calculated from Equations (3)–(5).The prediction metrics used to evaluate the abovementioned algorithms were RIn the field of spectroscopy, the RPD has been used as the standard method to report the quality of a model ,89,90,91In this study, correlation estimation models between hyperspectral data and photosynthetic characteristic parameters (Pn and FAPAR) were established using machine learning algorithms under different nitrogen conditions. The results showed that hyperspectral reflectance data and machine learning methods have excellent potential for estimating leaf Pn and canopy FAPAR in cotton. Among them, the LightGBM model , based on the hyperspectral first derivative, can better invert the Pn of cotton leaves. The RF model , based on the hyperspectral first derivative, can better invert the FAPAR of the cotton canopy. These results provide advanced metrics for non–destructive tracking of crop nitrogen status. In the future, these techniques can be used to diagnose nitrogen nutrition and the growth status of crops on large farms in real time.


Plant litter decomposition is a key process in carbon and nutrient cycling. Among the factors determining litter decomposition rates, the role of soil biota in the decomposition of different plant litter types and its modification by variations in climatic conditions is not well understood.In this study, we used litterbags with different mesh sizes  to investigate the effect of microorganisms and decomposer microarthropods on leaf and root litter decomposition along an altitudinal gradient of tropical montane rainforests in Ecuador. We examined decomposition rates, litter C and N concentrations, microbial biomass and activity, as well as decomposer microarthropod abundance over one year of exposure at three different altitudes .Leaf litter mass loss did not differ between the 1,000 and 2,000 m sites, while root litter mass loss decreased with increasing altitude. Changes in microbial biomass and activity paralleled the changes in litter decomposition rates. Access of microarthropods to litterbags only increased root litter mass loss significantly at 3,000 m. The results suggest that the impacts of climatic conditions differentially affect the decomposition of leaf and root litter, and these modifications are modulated by the quality of the local litter material. The findings also highlight litter quality as the dominant force structuring detritivore communities. Overall, the results support the view that microorganisms mostly drive decomposition processes in tropical montane rainforests with soil microarthropods playing a more important role in decomposing low-quality litter material. Dead leaves and roots comprise the major plant litter material that enters the belowground system and represent the main energy resource for soil organisms . AlthougLeaf and root litter differ in structure and chemical composition . GeneralEffects of soil organisms on litter decomposition not only change with litter traits, but also with climatic conditions . In tropvia litter consumption or through stimulation of microbial activity and transport of microbial propagules  the decomposition of litter decreases with increasing altitude, and this is mainly due to less favourable abiotic conditions. We also hypothesized that (2) access of litter by soil microarthropods increases decomposition as well as microbial biomass and activity in both leaf and root litter. Further, we hypothesized that (3) the abundance of decomposer microarthropods and Oribatida species richness increase with increasing microbial biomass in both leaf and root litter. Lastly, we hypothesized that (4) increase in litter quality (as indicated by litter C-to-N ratio) and microbial biomass during decomposition are key factors structuring Oribatida communities in both leaf and root litter.−1 at the respective sites , medium (1–2 mm diameter) and large (>3 mm diameter). The amount of individual leaf species and root size classes placed in the litterbags was chosen to resemble their amount in the litter layer and soil, respectively (see Nylon litterbags (17 × 17 cm) with mesh sizes of 45 µm, 1 mm and 4 mm were filled with 10 g of leaf or root litter. The mesh sizes we used are typically used to evaluate the role of meso- and macrofauna in decomposition processes. The 45 µm mesh prevents access by even the smallest mites and springtails, the 1 mm mesh allows access by virtually all mesofauna species and the 4 mm mesh allows access by virtually all macrofauna species . Leaf lively see . This waLitterbags were placed in the field in October 2008 (end of the rainy season). Bags containing leaf litter were randomly placed on top of the litter layer and fixed with nails, while those containing root litter were placed approximately 5 cm below the litter layer. Three blocks were established at each of the three altitudes with a minimum distance between blocks of 20 m. Two replicates of each treatment were placed in each block, with one replicate retrieved after 6 months and the other after 12 months.After retrieval, the litter material in each litterbag was divided into two parts of equal mass. The first half was analyzed for dry mass, microbial biomass, basal respiration, and C and N concentrations. From the second half, Oribatida and Collembola were extracted using a modified high-gradient heat extractor  and counloss) for both leaves and roots were calculated as Mloss = ((m0 − m1/m0)) × 100, where m0 is the dry weight of the initial litter placed in the litterbags and m1 the dry weight of litter at harvest. To measure carbon (C) and nitrogen (N) concentrations, dried  leaves and roots were milled to powder (<1 mm) and analyzed using a CN elemental analyzer .Mass loss (Mmic) were measured using a computer-controlled O2 micro-compensation apparatus (2 g−1 dry weight h−1) was determined as mean O2 consumption rates 10 to 20 h after attachment of the samples to the respirometer. Cmic was calculated from the maximum initial respiratory response  measured after glucose saturation following the substrate-induced respiration method (SIR) of mic was calculated as Cmic = 38 × MIRR (mg g−1 dry weight)  and microbial biomass (Cpparatus . BR (µl  weight) .loss, Cmic , BR, the abundance of Oribatida and Collembola, and Oribatida species richness data separately for leaf and root litter (hypotheses 1 and 2). Sampling date (6 and 12 months), mesh size , altitude  and all possible interactions were fitted as fixed factors, and block was fitted as a random factor with random intercept .Analyses were performed using R version 3.6.0 . Linear loss, C-to-N ratio) as well as microbial indicators  (hypothesis 4). Only species with more than three individuals in the samples were included. Monte Carlo randomization tests using 999 simulations were used to determine the significance of the axes. Sampling date (6 and 12 months), mesh sizes  and altitude  were coded as supplementary variables not affecting the ordination. Since the global test with all litter and microbial indicators was significant, we used forward selection to identify the most important variables structuring Oribatida communities. This was done to reduce the number of explanatory variables entering the analysis while keeping the variation explained caused by them at a maximum. The forward selection procedure was stopped if a variable reached a level of significance > 0.05 and R2 > 0.90  performed in CANOCO 5.02  was used2 > 0.90 .loss significantly increased during the time of exposure in both leaf and root litter. Leaf litter Mloss was not significantly affected by any interaction between the three factors studied, but it was higher at 1,000 and 2,000 m compared to 3,000 m at both sampling dates , Mloss for 4.3%  and BR for 3.9% . The community composition of Oribatida at 2,000 and 3,000 m correlated positively with increasing litter C-to-N ratio and BR; Mloss correlated positively with the first sampling date and was associated with lower species abundance.In total, 176 species of Oribatida were identified  and BR for 3.2% . As in leaf litter, Oribatida species at 2,000 and 3,000 m correlated positively with increasing litter C-to-N ratio, but BR correlated positively with the sampling date after 12 months. In both the CCA of leaf and root litter, the 1,000 m site was separated from the 2,000 and 3,000 m sites along the first axis and the centroids of mesh size were close to the center of the ordination. As note of caution, however, most Oribatida species were rare and only occurred at certain altitudes and at specific sampling dates; for the abundance of species at each of the study sites see CCA of Oribatida species in root litter with litter C-to-N ratio, BR, Mposition ; litter loss of both leaf and root litter showed different patterns of decomposition along the altitudinal gradient. For leaf litter, Mloss did not follow the expected linear decrease with altitude, rather, decomposition rates at 1,000 and 2,000 m were similar after the 12 months of exposure. This contrasts previous studies at our study sites . The contrasting results between leaf and root litter decomposition rates suggest that in the studied tropical montane rainforests differences in litter chemical composition and their association with nutrient limitations among the altitudinal sites are more critical factors for the decomposition of root litter than for leaf litter. Nonetheless, a complete litter chemical composition profile is needed to support this conclusion. Moreover, leaf litter might be more susceptible than roots to effects of climatic variations as leaf litter is located on top of the soil and thereby exposed to more variable microclimatic conditions than root litter in soil (In contrast to leaf litter, root litter showed the expected linear decrease in litter decomposition with increasing altitude after 12 months. Less favorable abiotic conditions, such as those at higher altitude, might affect root litter decomposition by reducing the quality of the litter material and thereby nutrient availability as reported for tropical rainforests . This is in soil . Howeverloss, Cmic and BR in leaf litter. Both decomposer groups may play a more important role at more advanced stages of decomposition when microorganisms have colonized leaf litter making it more palatable for arthropod consumers (loss at 3,000 m in 4 mm mesh bags suggests that under unfavourable environmental conditions, the decomposition of low-quality litter is stimulated by soil arthropods. Several processes may have accelerated root litter decomposition including stimulation of microbial growth via nutrient mobilization, litter fragmentation and dispersal of microbial propagules (mic and BR in roots increased in 1 and 4 mm mesh bags supports these findings and reinforces that the contribution of microarthropods to decomposition of recalcitrant substrates is more pronounced than in readily decomposable materials (The abundance of both Collembola and Oribatida was higher in 1 and 4 mm mesh bags irrespective of the plant litter type indicating that, as intended, 45 µm mesh size restricted the access to the litterbags by mesofauna. Thus, the different mesh sizes are a useful tool to evaluate the effects of decomposer animals on decomposition processes. However, restricting the access of mesofauna in 45 µm litterbags was more effective in Oribatida than in Collembola, indicating that the mesh size approach is limited for evaluating the role of mesofauna for decomposition processes and suggesting that it likely underestimates their effects on litter decomposition as discussed earlier . Furtheronsumers . Howeveropagules . The facaterials .mic and BR varied with mesh size in both leaf and root litter with the effect in root but not in leaf litter varying with altitude. Generally, Cmic was higher in 1 and 4 mm mesh bags, while BR was higher in 45 µm mesh bags. Overall, this supports our expectation that decomposer microarthropods stimulate microorganisms, thereby enhancing their biomass. Potentially, grazing on microorganisms results in increased nutrient mobilization favouring microbial growth (Notably, Cl growth . Furtherl growth . Indeed,l growth .loss, Cmic and BR. These results support our third hypothesis and suggest that litter accumulation in nutrient-poor sites can provide more habitat space for soil decomposer microarthropods. Notably, the increase in animal abundance and diversity may enhance the faunal contribution to litter decomposition (Additionally, despite the general lack of effect of mesh size on root litter mass loss, the abundances of both Collembola and Oribatida, and Oribatida species richness positively correlated with Mposition . Moreoveposition . Furtherposition . Therefoposition , soil arInterestingly, the abundance of both decomposer microarthropods in leaf and root litter did not vary significantly with sampling date. Potentially, the nutritional value of the litter material for these detritivore microarthropods changed little during the exposure time. Substrate quality may also be related to the generally low effects of soil decomposer microarthropods on leaf and root litter decomposition. Litter of different quality may attract different faunal communities and this likely contributes to the varying effects of decomposer animals on decomposition processes .Similar to Oribatida abundance, the higher Oribatida species richness in litterbags of 1 and 4 mm mesh size in both litter types might be attributed to restricted access of microarthropods to the 45 µm mesh litterbags. Oribatida species richness mostly varied with altitude in both leaf and root litter. The significant decrease in Oribatida species richness with increasing altitude in leaf litter supports results of previous studies at our study sites in that species richness of Oribatida in leaf litter is driven predominantly by factors linked to altitude such as temperature, precipitation, moisture and soil pH . By contSternoppia mirbilis, Lanceoppia sp.1 and Rostrozetes sp.6 preferentially colonized litter after 12 months of exposure with the latter two species exclusively recorded at 2,000 m. This supports our fourth hypothesis indicating that certain Oribatida species rely on food resources associated with changes in leaf litter physicochemistry during decomposition particularly at higher elevations, suggesting that Oribatida species diversity at least in part is due to resource partitioning (mic and BR with time were more pronounced in root than in leaf litter. This suggests that in contrast to our expectations, Oribatida community structure in root litter is not principally linked with decomposition or the gross characteristics of microbial communities. However, potential effects of root litter decomposition on the general quality of the organic litter material may function as key factor structuring Oribatida communities as indicated by their relationship with C-to-N ratios. Additionally, as mentioned before, root exudates may also modify the general characteristics of the organic matter and become an important carbon source fuelling Oribatida communities (mic , significantly correlated with Oribatida community composition and this is consistent with earlier findings (Similar to Oribatida species richness, Oribatida community assemblages varied mostly with altitude in both leaf and root litter. Most of the 176 species identified were registered on the 1,000 m site, only few species were only present at 2,000 and 3,000 m, presumably reflecting less favorable climatic conditions and poor resource quality at the highest altitudes . Interesitioning . This ismunities . Overallfindings .The results of our study suggest that the decomposition of both leaf and root litter in montane rainforests is mainly due to microorganisms when compared to the role of microarthropods. However, soil microarthropods may contribute to the decomposition of low-quality litter such as root litter, particularly at very high altitude. Generally, the contrasting patterns of microbial biomass and the abundance of decomposer microarthropods and Oribatida species richness with the time of exposure highlight the minor importance of microorganism abundance as a food resource and structuring force of decomposer microarthropod communities. Rather, the results point to the dominance of litter characteristics, such as litter C-to-N ratio, as key factor structuring Oribatida communities in both leaf and root litter. Additionally, our findings highlight the main role of climatic factors driving root litter decomposition across altitudinal gradients, but their effects on leaf litter decomposition might be overridden at the local scale by litter traits and biotic factors. Further, at local scales differences in litter characteristics and the nutritional requirements of decomposer communities may modulate both leaf and root litter decomposition, and nutrient cycling in tropical montane rainforest ecosystem. Importantly, however, our 12 months study may not have been long enough to uncover the total contribution of soil microarthropods to litter decomposition. Due to general low quality of the litter material in this tropical rainforest, the functional role of soil microarthropods might become more significant in the long-term. Nonetheless, the study contributed to a better understanding of the role of decomposer microarthropods for litter decomposition in tropical rainforest ecosystems.10.7717/peerj.14264/supp-1Data S1Click here for additional data file.10.7717/peerj.14264/supp-2Supplemental Information 2Click here for additional data file.


There is persistent coronary vasomotor tone during myocardial ischemia, despite ongoing coronary arteriolar dilatation. The mechanism underlying this vasodilatory tone, which can be unmasked by coronary vasodilators, is unclear. We hypothesized that persistent microvascular resistance during myocardial ischemia occurs at the level of capillaries and may be caused by pericytes.We studied nine instrumented dogs where coronary blood flow and coronary driving pressure were reduced to half by placement of stenoses. Myocardial blood flow and myocardial blood volume were measured with myocardial contrast echocardiography before and during adenosine administration. In three animals, the heart was perfusion-fixed under these conditions for electron microscopic assessment of capillary and pericyte size.During ischemia, myocardial blood volume decreased and myocardial vascular resistance remained unchanged. Adenosine administration reversed the decline in myocardial blood volume and decreased myocardial vascular resistance. Electron microscopy showed larger capillaries in ischemic beds receiving adenosine than ischemic beds not receiving adenosine. Pericytes in beds receiving adenosine also tended to be larger.Capillaries are the site of persistent vasomotor tone during myocardial ischemia; any other site of vascular regulation (arterioles or venules) cannot explain these myocardial contrast echocardiography findings, which are confirmed on post-mortem electron microscopic examination. The decrease in capillary size is likely caused by pericyte contraction in an attempt to maintain a constant capillary hydrostatic pressure. Adenosine relaxes pericytes, restores myocardial blood volume, reduces myocardial vascular resistance, and improves regional function during ischemia. These findings could have important therapeutic implications. Several investigators have described the presence of residual coronary vasomotor tone during myocardial ischemia –5. This Using myocardial contrast echocardiography (MCE), we previously showed that when CBF decreases moderately at rest, myocardial blood volume (MBV), 90% of which resides in myocardial capillaries, decreases, while total microvascular resistance (MVR) remains unchanged . At veryAlthough pericytes were described almost 150 years ago , differein vivo that when perfusion pressure decreases distal to a critical femoral artery stenosis during hyperemia, pericytes contract, resulting in capillary de-recruitment . Adenosine has been shown to relax pericytes by activating ATP-sensitive K+ channels –25. On e−1) and maintain (200 mg.hr−1) anesthesia throughout the experiment. The heart rate, PO2, end-tidal CO2, and temperature were continuously monitored . Catheters (7F) were placed in both the femoral veins for microbubble infusion and administration of fluids and drugs, respectively.The study was approved by the Animal Care and Use Committee at Oregon Health & Science University and conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. In total, nine adult male dogs (25–35 Kg) were studied. Female animals were not used to avoid potential for undetected pregnancy. The animals were intubated and ventilated with room air. Sodium pentobarbital was used to induce  were inserted into the right atrium (RA) and retrogradely into the descending aorta to measure pressure. The proximal portions of the left anterior descending (LAD) and left circumflex (LCx) coronary arteries were dissected free from surrounding tissue. Time-of-flight ultrasonic flow probes  were placed on proximal portions of both arteries and connected to a digital flow meter  to monitor and record CBF. A custom-designed occluder was placed on both coronary arteries distal to the flow probes . PolyethCatheters and flow meters were interfaced to a multi-channel recorder . CBF and pressures in both coronary arteries, as well as mean central aortic and right atrial pressures, were acquired digitally and displayed on-line on a computer system . All data were saved and analyzed off-line using LabChart 6.Coronary venous blood lactate and pH were measured in the blood samples (0.8 mL in 1.0 mL heparinized syringes) obtained from both coronary veins. The samples were immediately placed on ice and analyzed within 15 min using an ABL800-FLEX analyzer .Two-dimensional echocardiography was performed using a Sonos-5500 System  interfaced to a phased-array S-3 transducer capable of harmonic imaging. The transducer was affixed to the procedure table using a custom-designed arm. A saline bath acted as an interface between the transducer and the heart. Ultrasound gel was placed between the bath and the anterior cardiac surface. For regional wall thickening (WT) measurements, real-time images were acquired at the mid-papillary muscle short-axis level and stored for later analysis.Our method for WT quantification has been previously described . Briefly®  infused at 150 mL·hr−1. After steady state is achieved, this concentration of microbubbles produces adequate myocardial opacification without posterior wall attenuation. Images were acquired with ultrasound gated to the electrocardiogram at end-systole at pulsing intervals (PIs) of 1–3, 5, 8, 10, and 20 cardiac cycles to allow for progressively greater bubble replenishment of the ultrasound beam elevation , where y = acoustic density at PI t, A = acoustic density after the ultrasound beam is completely replenished (MBV fraction), and β = rate constant that reflects the mean myocardial blood flow velocity. The product, A•β, was calculated to derive regional MBF . The samples were rinsed three times with sodium cacodylate buffer for 40 s at 150 W, stained with 2% osmium tetroxide and 1.5% potassium ferricyanide for 13 min with a cycle of 3 min on at 100 W and 2 min off, rinsed with water three times for 40 s at 150 W, stained en bloc with uranyl acetate for 6 min at 100 W with a cycle of 2 min on, 2 min off, dehydrated in an acetone series for 40 s at 150 W, infiltrated with resin for 15 min at 150 W, and polymerized at 60 °C. The sections, 70 nm thick, were post-stained with uranyl acetate and lead citrate and imaged at 120 kV in an FEI Tecnai T12  transmission electron microscope equipped with a NanoSprint 12  camera. Images of the heart were used to quantify the capillary circumference and diameter . The size of pericytes and their processes surrounding the capillaries was also planimetered.The heart tissue from each vascular bed was separated into endocardial, mid-wall, and epicardial one-thirds. Each section was then divided into multiple pieces measuring about 1 mmin vivo using 0.1 mL of Lumason® diluted to 3 mL with normal saline and injected sub-selectively into either artery. A primary stenosis was created on either an LAD or LCx to reduce CBF to below 50% of baseline, and all data were collected. Adenosine was then infused into the artery (5 μg·kg−1·min−1 at a rate of 3 mL·min−1). Four min into infusion, data collection was initiated after completion of which adenosine infusion was discontinued. A secondary flow reducing stenosis was then placed on the other artery (positive control), and all data were collected again.All baseline hemodynamic, CBF, 2-dimensional echocardiography, and MCE data were first obtained, followed by blood collection from veins. This sequence of data collection was repeated at each stage of the experiment. CBF and CDP for each artery were initially measured after 30 s of total coronary occlusion to define maximal hyperemic CBF. The LAD and LCx perfusion bed sizes were then defined After completion of the aforementioned stages, adenosine infusion was restarted in the artery that had previously received it. In three dogs, a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, was infused in both arteries at the same rate for EM studies. After 10 min of infusion, the animals were euthanized with a mixture of pentobarbital and KCl, and the hearts were removed and cut into 5 short-axis slices. The slice corresponding to the MCE images (defined by placing a needle through the heart at the level of the ultrasound transducer) was processed for EM. The LAD and LCx beds were defined based on MCE images obtained during intracoronary injection of microbubbles.t-test. A modified Bonferroni correction was used for multiple comparisons. For differences between groups, a p-value of <0.05 (two-sided) was considered significant.Data are expressed as mean ± 1 SEM. Comparisons between stages were performed using either ANOVA or unpaired Student's −1 and 94 ± 13 mL·min−1 for the LAD and 36 ± 4 mL·min−1 and 79 ± 13 mL·min−1 for the LCx, respectively. Primary stenoses (those examined before and after adenosine) were created in nine animals. One animal died after creation of the primary stenosis; hence, secondary stenoses were created in eight animals.CBF at rest and maximal hyperemia (mean ± 1SEM) were 35 ± 4 mL·minn = 5) of complete blood collections. Importantly, the distal coronary pressure was similar after creation of both stenoses.During adenosine infusion into the coronary artery with the primary stenosis heart rate, mean aortic pressure and the rate pressure product remained unchanged . CBF incA) decreased after placement of both stenoses as did MBF velocity (β) and MCE-derived MBF. The decreases were quite similar for both the primary and secondary stenoses, confirming that the secondary stenosis was an adequate positive control. During adenosine infusion, MBV (A) in the bed supplied by the primary stenosis increased significantly, but MBF velocity (β) did not change. All the increase in MCE-derived MBF was therefore a consequence of the increase in MBV (A). There were no significant changes in MCE parameters in the bed supplied by the secondary stenosis (defined on MCE by injecting microbubbles in the artery where stenosis was subsequently placed) when adenosine was infused into the bed with primary stenosis  during reduced CDP underlies the persistent microvascular tone seen during myocardial ischemia, since it reverses with adenosine administration. Thus, capillaries, which comprise 90% of MBV on MCE, are the site of persistent microvascular tone during myocardial ischemia; any other site of vascular regulation (arterioles or venules) cannot explain these findings because arterioles and venules contribute minimally to MBV measured on MCE during systole  remains the same or even increases –12: the Tissue homeostasis is essential for cell health and is maintained by the hydrostatic and oncotic pressures in the capillary and interstitium, respectively, as described by Starling 125 years ago . Of thesin vivo neural/glial antigen 2 mouse hind limb preparation where pericytes appear red, we recently demonstrated that when perfusion pressure drops beyond a critical femoral artery stenosis during exogenous hyperemia, capillaries constrict at site of pericyte locations, and this finding is significantly attenuated in mice with partial pericyte depletion  and an increase in MVR during reduced CDP underlie the persistent microvascular tone seen during myocardial ischemia, since these findings reverse with adenosine administration. These findings indicate that capillaries are the site of persistent microvascular tone during myocardial ischemia. Any other site of vascular regulation (arterioles or venules) cannot result in this combined effect of decreased MBV and increased MVR. These findings are confirmed on post-mortem EM examination of capillary size. We provide circumstantial evidence to indicate that pericyte contraction may be responsible for the reduction in MBV. Direct in vivo observations will be required to confirm these findings, which could have important clinical implications for the treatment of myocardial ischemia.In summary, a decrease in MBV (measured The raw data supporting the conclusions of this article will be made available by authors upon request, without undue reservation.The animal study was reviewed and approved by the Animal Care and Use Committee at Oregon Health & Science University.DL developed and performed the experiments, analyzed and tabulated the results, and drafted the manuscript. YZ assisted with the experiments. SK developed the hypothesis and study design, assisted with the experiments, reviewed the data, and helped with manuscript development. All authors contributed to the article and approved the submitted version.This research was supported by the Garthe and Grace L. Brown Fund of the Oregon Community Foundation.The contents do not represent the views of the U.S. Department of Veterans Affairs or the U.S. government.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.