However, groups with the association in cyanoacrylate showed superior healing, and analgesic action to the isolated cyanoacrylate group. In addition, two studies demonstrated that cyanoacrylate use reduces surgery duration, one study showed that it reduces postoperative sensibility, and another present hemostatic effect (p < 0.05). There is scarce literature for the use of cyanoacrylate in FGG wounds indicates that it can promote a minor inflammatory response, reduce operation time, does not interfere with healing, relieves postoperative discomfort, and suggests the possibility immediate hemostasis. Its use presents an alternative to suturing in FGG surgeries. But, the limited number of cases and the relative heterogeneity of the included studies suggest caution in generalizing the indication.To analyze the hemostatic, Dsurgical wounds in donor and recipient areas of free gingival grafts (FGG). Five databases  were searched up to March 2021 (PROSPERO CRD42019134497). The focus of the study (cyanoacrylate) was combined with the condition , and outcome . Studies reporting cyanoacrylate isolated or associated with another substance in FGG stabilization and closure were investigated and assessed for the quality and risk of bias through the Cochrane Manual. Six studies with 323 participants were included. Evaluation of the quality and risk of bias highlighted a low risk for four articles, intermediate for one and unclear for another. The use of cyanoacrylate associated or not with the hemostatic sponge or the platelet-rich fibrin was more effective in healing (three studies), analgesia (four studies), and hemostasis in one study (Scientific justification: To evaluate the hemostatic, analgesic and healing actions of cyanoacrylate compared to the suture thread and other agents when used to close surgical wounds from periodontal free gingival graft surgical wounds in both the donor and recipient areas of the graft. Main findings: The use of cyanoacrylate individually or in association with wound dressing agents presents analgesic effects because the patient reports less pain experienced when cyanoacrylate is applied to the wound closure and covering, thereby reducing the need for postoperative analgesic medication. In addition, a healing effect is observed in the closure of the donor area on the palate; as well as it seems to present hemostatic effects, reducing the bleeding time after surgery, and preventing late bleeding during the first postsurgical week. Practical implications: Dentists may cautiously apply cyanoacrylate after periodontal surgeries for free gingival graft in both the donor and recipient areas of the graft. However, they must consider the limitations of the surgery, tension-free positioning, the patient’s dyscrasia and postoperative care, constituting a set of predictors for adequate clinical decision-making. Widespread use of such material for all patients and surgical configurations may not be recommended.Cyanoacrylate seems to present analgesic effects and less pain when applied to wound closure and covering donor and recipient areas reducing the need for postoperative analgesic medication; and has a healing effect in the closure of the donor area on the palate. In addition, it can reduce bleeding time after surgery, and prevents late bleeding during the first postsurgical week.  The choice of material to establish good tissue synthesis is extremely important for postoperative success. Tissue synthesis aims to maintain the tissues well coaptated in order to accelerate the healing process, prevent bleeding and contribute to forming and maintaining the blood clot, thereby avoiding infection at the site, contamination of the surgical wound and postoperative pain , 2.Cyanoacrylate adhesives have been extensively used for closing skin wounds and in several surgical procedures involving skin, mucous membranes and different tissues, including oral tissues –5. TheseThe main advantages of bioadhesive materials are generally attributed to the following factors: high tissue compatibility and long half-life; presence of hemostatic, analgesic and antibacterial properties; high potential for adhesiveness and biodegradation; the ability to maintain the position/stabilization of injured tissues; and to trigger a mild inflammatory response , 4, 6, 9In periodontal surgeries, when the free gingival graft is used to increase the range of keratinized mucosa, the donor tissue thickness and the graft stabilization in the recipient area are vital to protect local vessels against damage and dehydration, thereby decreasing the possibility of bleeding, tissue retraction, and consequently contamination of the surgical wound and postoperative pain . It is pIn this context, the objective of this systematic review was to analyze the hemostatic, analgesic and healing effects of cyanoacrylate when applied to surgical wounds in donor and recipient areas of free gingival grafts (FGG), and compare them with those produced using conventional sutures and other materials and techniques.https://www.crd.york.ac.uk/PROSPERO/display_record.php? RecordID=134497). This systematic review addressed a clearly focused issue, adopting the population, intervention, comparison and outcome (PICO) method  dp < 0.01), and exhibited the lowest consumption of analgesics (10%) compared to the control group (50%). In other study, Tavelli et al. (2018) [p < 0.05). The isolated cyanoacrylate group had a numerically lower perception of pain compared to the suture group. The cyanoacrylate associated hemostatic sponge group (10%) and isolated cyanoacrylate (20%) groups showed the lowest analgesic consumption. Stavropoulou et al. (2019) [For Tavelli et al. 2019) [ [9], the. (2018) , showed . (2019)  showed talgesics % compare. (2019) , 14. Heaalgesics % comparealgesics % comparep < 0.05) in their study, meaning better tissue healing than the other two groups  at 6 months follow-up. In the study by Ozcan et al. (2017) [Gümüş & Buduneli 2014) [4 [1] usep < 0.001). In addition, cyanoacrylate bioadhesive group and DLP (Double-layered Protection with cyanoacrylate and hemostatic gelatin sponge) group promoted better healing compared to the control group with suturing only (p < 0.001).Finally, Tavelli et al., 2018  used theThe results of the present systematic review indicate that the use of cyanoacrylate associated or not with the hemostatic sponge or the PRF was more effective in hemostasis, healing, and analgesia compared to the control group, however on the basis of relatively limited clinical evidence. Furthermore, groups with the cyanoacrylate in association showed superior effects than the isolated cyanoacrylate group.Free gingival graft (FGG) is a widely used surgical procedure to increase the dimensions of the inserted gingiva. Its autogenous character, maintenance of tissue keratinization, predictability of the results and the ease of the technique have made FGG considered as the gold standard among gingival augmentation procedures , 16. Howp < 0.05). This demonstrates that the use of cyanoacrylate to close the free gingival graft surgical wound can optimize professionals’ and patients’ time. Furthermore, these authors related this shorter operating time to reduced inflammation and consequently edema and pain in the postoperative period.Cyanoacrylate stands out among the materials on the market for the closure of surgical wounds which has been pointed out by different authors , 3, 4, 6Cyanoacrylate can be used in several oral surgical procedures with clinical and histological activity, as it has been shown to work as an excellent surgical cement and hemostatic agent which is well tolerated by the tissues, thus facilitating the healing process and also reduces the surgical time –21. The Considering that cyanoacrylate seals the edges of the surgical wound acting as a superficial plug without allowing space for fluids or other oral products to interfere during healing, thus isolating the wound margins from the actions of saliva, food debris and biofilm , it is pIn the case of FGG, the tissue contraction usually resulting from the healing process seems to occur in two phases: first, during the formation of a network of blood vessels in the graft, and soon after when the grafts integrate with the recipient area . The grap < 0.05) or better epithelialization, meaning better tissue healing than the other test groups. The divergence in results between these studies can be attributed to the different methodologies used, and not only involving the investigated groups, but also the follow-up times and the strategy of evaluation.Two studies , 14 diffThe literature , 19, 23 The hemostatic potential of cyanoacrylate in preventing bleeding, associated or not with coagulation disorders in oral surgery, has been previously evaluated , 27–30. Cyanoacrylate was widely accepted by patients when used as a protector for the graft donor region due to pain relief and reduced discomfort during feeding . The abiCyanoacrylate has been used in several oral surgical procedures, including surgeries for FGG, in which it has been shown to be comparable or even superior to suturing due to its greater ease of use, reduced operative time, immediate hemostasis production and for promoting postoperative comfort , 13, 18.The included articles were generally considered to have a low risk of bias, as there was no high risk of bias classification in any of the items assessed in the articles included in this review. The items considered “unclear risk of bias” fell into this category when the parameter was reported, but the precise execution was not clear. As an example, the item “Generation of the random sequence” was classified as unclear risk of bias in the article by Barbosa et al. (2009)  because,Heterogeneity in the free gingival graft recipient area was observed in the included studies; however, the graft donor area was the same in all studies, namely: the posterior palatal mucosa, usually involving the region between the first premolar and the maxillary first molar. Only one study evaluated the effect of applying or not applying cyanoacrylate on a hemostatic sponge, but it did not have an exclusive cyanoacrylate group. In addition, this review did not assess the effect on clinical the increase in keratinized tissue and includes studies which use cyanoacrylate for different indications, thus making comparisons difficult. Given the heterogeneity of the outcomes, no Meta-analysis was performed.n = 323) suggest considerable caution when interpreting the results and highlight the need for more properly designed clinical trials.The cyanoacrylate-based adhesives (either exclusive or associated) generated; less perception and experience of pain, reduced postoperative discomfort and lowered analgesic consumption; did not interfere in the healing process, presented lower graft shrinkage, and promoted a better epithelialization response compared with suturing. In addition, cyanoacrylate promotes reduced surgical time, including reducing the number of follow-up visits as suture removal becomes unnecessary. These characteristics of cyanoacrylate suggest its exclusive use or associated with other techniques and substances as an alternative to conventional suturing in free gingival graft surgeries in both the donor and recipient areas. The hemostatic activity could not be confirmed because only one study presented this information. However, relative methodological limitations of the selected studies and the total number of study subjects (


NOTCH2NLC gene has been identified as the etiopathological agent of NIID. Interestingly, this GGC repeat expansion was also reported in some patients with a clinical diagnosis of amyotrophic lateral sclerosis (ALS). However, there are no autopsy-confirmed cases of concurrent NIID and ALS.Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease. Pathologically, it is characterized by eosinophilic hyaline intranuclear inclusions in the cells of the visceral organs as well as central, peripheral, and autonomic nervous system cells. Recently, a GGC repeat expansion in the A 60-year-old Taiwanese woman reported a four-month history of progressive weakness beginning in the right foot that spread to all four extremities. She was diagnosed with ALS because she met the revised El Escorial diagnostic criteria for definite ALS with upper and lower motor neuron involvement in the cervical, thoracic, and lumbosacral regions. She died of respiratory failure at 22 months from ALS onset, at the age of 62 years. Brain magnetic resonance imaging (MRI) revealed lesions in the medial part of the cerebellar hemisphere, right beside the vermis . The subclinical neuropathy, indicated by a nerve conduction study (NCS), prompted a potential diagnosis of NIID. Antemortem skin biopsy and autopsy confirmed the coexistence of pathology consistent with both ALS and NIID. We observed neither eccentric distribution of p62-positive intranuclear inclusions in the areas with abundant large motor neurons nor cytopathological coexistence of ALS and NIID pathology in motor neurons. This finding suggested that ALS and NIID developed independently in this patient.We describe a case of concurrent NIID and ALS discovered during an autopsy. Abnormal brain MRI findings, including paravermal lesions, could indicate the coexistence of NIID even in patients with ALS showing characteristic clinical phenotypes. Neuronal intranuclear inclusion disease (NIID), a rare neurodegenerative disease, is pathologically characterized by eosinophilic hyaline intranuclear inclusions in the cells of the visceral organs as well as central, peripheral, and autonomic nervous system cells , 2. NIIDNOTCH2NLC gene as NIID’s underlying etiopathology. This abnormal GGC repeat expansion in the NOTCH2NLC gene is reportedly associated with essential tremor and leukoencephalopathy [NOTCH2NLC gene has appeared in 4 out of 545 patients with a clinical diagnosis of amyotrophic lateral sclerosis (ALS) [Recently, techniques such as long-read sequencing , 4 and dalopathy , 7. Moreis (ALS) . These oHere, we describe a case wherein the main clinical features were consistent with ALS diagnosis, but an autopsy confirmed the coexistence of both NIID and ALS.A 60-year-old Taiwanese woman presented to the Chiba University Hospital with a four-month history of progressive weakness beginning in the right foot and spreading to all four extremities. Her medical history was unremarkable except for cough-variant asthma, and there was no family history of neuromuscular or neurodegenerative disorders.A neurological examination showed no abnormality of the cranial nerves except for slight dysarthria. However, reduced muscle strength in all four extremities, especially in the right foot, with wasting, was observed. Fasciculations were noted in both arms. Deep tendon reflexes were brisk with a positive Trömner sign on the right, and bilateral plantar reflexes were extensor. Abdominal skin reflexes were absent bilaterally, whereas abdominal muscle reflex was enhanced. There were no signs of cerebellar dysfunction. The sensation was also intact to all modalities used for testing. The patient scored 28/30 on the Mini-Mental State Examination (MMSE) as she lost points for recall and following a command. She scored 16/18 on the Frontal Assessment Battery (FAB) where she lost points for similarities and lexical fluency. The overall score of Addenbrooke’s Cognitive Examination III was 83 out of 100, with subscores of 18/18 for attention and orientation, 15/26 for memory, 9/14 for fluency, 25/26 for language, and 16/16 for visuospatial skills.Needle electromyography demonstrated active and chronic denervation potentials in her biceps brachii, first dorsal interosseous, thoracic paraspinal muscles, tibialis anterior, and vastus lateralis. Nerve conduction studies (NCS) in the right upper and lower limbs revealed reduced conduction velocities and prolonged distal latencies in all tested motor nerves Table . DecreasThe patient met the El Escorial diagnostic criteria for definite ALS with upper and lower motor neuron involvement in the cervical, thoracic, and lumbosacral regions . On the Subsequently, clinical signs of upper and lower motor neuron involvement in the bulbar lesion became evident. The patient underwent a gastrostomy eight months after ALS onset and died 22 months after onset, at 62 years of age, due to respiratory failure.The patient’s family provided informed consent for postmortem analyses, and an autopsy was performed. Appropriate brain areas were dissected and embedded in paraffin, 5 μm-thick serial sections were stained with hematoxylin and eosin and Klüver-Barrera (KB), and then immunostained against phosphorylated transactivation response DNA-binding protein (TARDBP) 43 kDa (pTDP-43) , TDP-43  and p62 .At autopsy, the brain weighed 1,020 g. The pia mater, particularly on the central regions’ surface, appeared cloudy, suggesting cortical atrophy in these areas Fig. . The preWe observed abundant p62-positive intranuclear inclusions in the cerebellar glial cells, particularly in the Purkinje and the granular cell layers and in the periventricular ependymal region Fig. . InclusiThe patient presented with a clinical course consistent with limb-onset ALS and met the El Escorial diagnostic criteria for a definite antemortem ALS diagnosis. Paravermal lesions in the medial part of the cerebellar hemisphere as seen in the brain MRI prompted a diagnosis of NIID, and both antemortem skin biopsy and postmortem autopsy confirmed the coexistence of ALS and NIID. Further, the absence of an eccentric distribution of p62-positive intranuclear inclusions in areas with abundant large motor neurons and no evidence of cytopathological coexistence of ALS and NIID pathology in the motor neurons suggests that ALS and NIID developed independently in this patient.NOTCH2NLC gene, which is etiopathological of NIID, has been observed in 4 out of 545 patients with a clinical diagnosis of ALS [NOTCH2NLC gene for this case; nevertheless, the autopsy confirmed the coexistence of pathology consistent with both ALS and NIID. Moreover, the distribution of p62-positive intranuclear inclusions and double immunohistochemical staining against p62 and TDP-43 suggested that ALS and NIID had developed independently in our patient. While ALS and NIID’s coincidental coexistence cannot be completely ruled out, the very low prevalence of these two conditions precludes any assessment of the probability of such an occurrence.ALS and NIID may share underlying etiologic factors but coexist independently in an individual. Intranuclear inclusions positive for ubiquitin and p62 have been reported in both ALS and NIID , 2, 10. s of ALS . While ts of ALS . UnfortuIt is possible that paravermal lesions in the medial part of the cerebellar hemisphere are the sole radiological clue for diagnosing NIID because, in our case, although the brain MRI revealed abnormal signals in the paravermal areas, findings typical of NIID, such as high-intensity signals along the corticomedullary junction on diffusion-weighted imaging (DWI) or diffuse high-intensity signals in cerebral white matter on FLAIR , 11, werThe results of NCS in our case may be interpreted as a mixture of NIID-associated subclinical peripheral neuropathy and ALS-related changes. Motor and peripheral sensory neuropathy is a common symptom in patients with NIID  and can NOTCH2NLC gene and CGG repeat length of the FMR1 gene could not be determined. Similarities in clinical, radiological, and pathological findings between NIID and fragile X- associated tremor/ataxia syndrome (FXTAS) have been reported [This case report’s limitations are that the GGC repeat length of the reported , 20, andreported . Howeverreported , and thereported . Second,reported , 24.In conclusion, we describe a case of coexisting NIID and ALS diagnosed at autopsy. Abnormal brain MRI findings, including paravermal lesions, could indicate the coexistence of NIID even in patients with ALS showing a typical clinical phenotype.


It is found that cell orientation is influenced by specific combinations of stiffness value and stiffness gradient. The results highlight the importance of considering the type of hydrogel as well as both the absolute value of the stiffness and the steepness of its gradient, thus introducing a new framework for the development of tissue engineered scaffolds and the study of substrate stiffness.Understanding the influence of the mechanical environment on neurite behavior is crucial in the development of peripheral nerve repair solutions, and could help tissue engineers to direct and guide regeneration. In this study, a new protocol to fabricate physiologically relevant hydrogel substrates with controlled mechanical cues is proposed. These hydrogels allow the analysis of the relative effects of both the absolute stiffness value and the local stiffness gradient on neural cell behavior, particularly for low stiffness values (1–2 kPa). NG108‐15 neural cell behavior is studied using well‐characterized collagen gradient substrates with stiffness values ranging from 1 to 10 kPa and gradient slopes of either 0.84 or 7.9 kPa mm A new protocol to fabricate a reproducible and mechanically characterized collagen based model of stiffness gradients, valuable for the study of durotaxis, is demonstrated. Morphological responses of neural cells under defined stiffness gradient profiles are investigated and for the first time the combined influence of both local stiffness magnitude and the gradient steepness is highlighted. This contributes significantly to the understanding of neural cell behavior and will underpin future improvements to nerve repair strategies. The current clinical solution, the nerve graft, is often unsatisfactoryThe interaction between the tissue and the cells is a key factor in controlling the fate of the latter.In order to inform the development of appropriate substrates, key information about the effect of local stiffness gradients both in terms of the base stiffness value and steepness of the gradient needs to be obtained. In this study, model substrates were developed using collagen hydrogels in which defined gradients of stiffness were generated and characterized. Collagen type I is the major ECM component of the PNS and supports cellular nerve function from development to adulthood.22.1FigureFor this study, gradient moulds were designed using the CAD software AutoCAD, and 3D printed from polylactic acid (PLA) using an Ultimaker 2. Two different multiwell moulds were produced, each of which contained wells of the same dimensions as a standard 24‐well plate and therefore could be used to cast multiple equivalent collagen hydrogels simultaneously. The bottom surface of each mould well was shaped to include 3× equally spaced raised ridges, conferring a pattern of varying depth on the collagen hydrogels cast in the moulds subsequently  was mixed at a ratio of 80%: 10 v/v% with 10 × Minimum Essential Medium (MEM), 5 v/v% of Dulbecco's modified Eagle's medium (DMEM), and was neutralized with 5 v/v% of 0.325 m NaOH to achieve pH 7.4.2) for 10 min to allow gelation.Acid‐solubilized type I collagen from rat tail tendon . This step rapidly removed most of the fluid of the hydrogel through the top surface of the gel and resulted in gels with a flat top and bottom surface, but with gradients of collagen density resulting from the variation in gel height as a consequence of the ridges in the original casting mould  and the mass of collagen (mc in mg) was calculated for each of the three segmented regions  is the density of collagen.Stabilized gel heights were measured using an optical contact angle meter (OCAM) (KSV's CAM 2000). The images were extracted and analyszed on ImageJ. For accuracy, three measurements of the height of each gel were taken and averaged. Using the height measured, the volume post stabilization  was used to obtain 10 × 10 µm high‐resolution topography images (1024 × 1024 pixels) from each segment . Experiments were carried out using force mapping over 1 × 2.5 mm area, covering the segments I–II–III, at room temperature. The collected force curves were batch‐analyzed using JPK/SPM data processing software . A Hertz model was used for determining the Young's Modulus E (kPa), assuming a Poisson ration (ν) of 0.5.In order to quantify the effect of collagen concentration on mechanical properties of the material, a series of force mapping experiments were performed to yield a stiffness map using AFM‐force spectroscopy. Indentation measurements were performed using a JPK Nanowizard CellHesion 200 with motorized precision stage . Glass microspheres  were glued  to nonconductive silicon nitride and triangular tip‐less cantilevers  contained in 24‐well plates, in serum‐free cell culture medium.t = 48 h) in a humidified incubator (5% CO2 at 37°).NG108‐15  were trypsinized and 1 × 102.7Cultures were fixed with 4% paraformaldehyde overnight at 4 °C and permeabilized using 0.5% Triton X‐100 for 30 min. Nonspecific binding was blocked with 5% normal goat serum. Nuclei were labeled using Hoechst (1:1000). Mouse anti‐β‐III‐tubulin primary antibody  and dylight goat antimouse 488  were used to detect the neurites. Samples were rinsed using PBS) between each step. Fluorescence microscopy  was used to acquire images of NG108‐15 cells on the gels. Images were captured using a 10× lens, and were analyzed using ImageJ software .2.8Neurite response to collagen gradients was quantified using established measures2.9P values. Statistical significance was taken at P < 0.05.For the mechanical and in vitro experiments, statistical analysis was performed using JMP Pro 13 software . Data are presented as mean values ± standard deviation (SD). A Shapiro–Wilk test was used to test the normality of the distributions. The differences between the multiple groups were evaluated by performing a one‐way ANOVA test followed by a Tukey–Kramer post hoc test to obtain the multiple comparison 33.1−1, and yielded a Young's Modulus (E) ranging from 1.7 ± 0.3 to 2.0 ± 0.2 kPa . The three segments (I–II–III) within both Lower and Higher gradient were significantly different .We fabricated collagen substrates with stiffnesses of physiological relevance to the PNSms Table , the con3.2FigureLower, therefore the overall surface topography and roughness of the gels can be negligible and the gels can be considered as flat.AFM imaging was used to examine the topography of the collagen gels and investigate any surface modification induced by the process of fabricating gradients. 3.3−1), and Higher gradient (7.96 kPa mm−1) gels, for each segment area  compared to the segment IHigher of the Higher gradient (E ≈ 1.5 kPa), suggesting that NG108‐15 cells were responding differently to the local gradients present in these substrates which were otherwise mechanically equivalent in terms of absolute stiffness. This phenomenon can be observed FigureFigure Lower & Higher) and the Control, respectively, 1.7, 1.5, and 1.0 kPa  elongated preferentially up the gradient, toward stiffer regions. In contrast, on the stiffer segment of the Higher gradient (IIIHigher), neurites elongated preferentially down the gradient toward the softer region. Overall, NG108‐15 cells responded to the local mechanical environment conferred by the gradient gels, altering both branching behavior and orientation.Figure 4Our primary goal was to design a flat substrate with a characterized rigidity gradient to study cell responses to mechanical cues. We created physiologically relevant stiffness gradients within the range of modulus values reported previously for PNS tissueFigurePrevious studies to create 2D substrates with continuous stiffness gradients have used coated polyacrylamide gels (PAAm) gels as well as proteins such as fibrin and gelatin. The range of stiffnesses obtained vary over several orders of magnitude−1 gradient induces Schwann cell durotaxis. Our Lower gradient (0.85 kPa mm−1) therefore could potentially be used to modulate both Schwann cells and neural cells combined for the improvement of nerve guidance conduits.By contrast, the approach used here does not affect the microstructure of the collagen fibrils and the banding pattern is conserved after gel stabilization, offering a way to make gradients purely of collagen, in a simple, controlled, and reproducible way. Previous studies have reported the effects of similar substrate stiffness profiles for other types of cells . A similar phenomenon was reported by Flanagan et al.Lower, and IHigher), the percentage of cells presenting branching neurites was significantly greater on the Lower gradient. This observation highlights the potential of neuronal cells to be mechanosensitive to specific stiffness gradient slopes as opposed to only the absolute stiffness value of the substrate. Rosso et al.,Lower, and IHigher ≈1 kPa). However, on the stiffest area of the Lower gradient gel (IIILower ≈2 kPa), we observed a preferential orientation toward stiffer areas. A preferential orientation was also observed on the stiffest region (IIIHigher ≈10 kPa) toward softer areas. It is known that the growth cone detects guidance cues and has a major role in neurite orientation.This study provides the first evidence that neurite elongation can be influenced by a combination of substrate stiffness absolute value and the steepness of the gradient slope, suggesting that neurites have the potential to process a combination of mechanical guidance cues. Neurite branching is potentially an important parameter for the establishment of synaptic connections,2 and 104 Pa) compared to soft substrates (10 Pa). However, in their study, no significant differences were found on substrates with a stiffness greater than 102 Pa. The difference observed could potentially be explained by the difference in the cell type used for the study, the difference in the substrate composition, the absence of additional external cues , or the introduction of a stiffness gradient slope as previously hypothesized by Leach et al.Neurons on different part of the stiffness gradient gels did not exhibit significant differences in neurite length, proportion of cells presenting neurites, or number of neurite per cell (substrate stiffness from 1 to 10 kPa). This result is in contrast with Rosso et al.,5This study describes a technique to fabricate type I collagen gels with controlled and reproducible physical (density) and mechanical (stiffness) gradients, without damaging collagen fibril structure nor adding additional chemical modification. These gradient gels offer a valuable platform to study cell–substrate interactions. Neural cells responded to the gradients by modifying their branching behavior and orientation. This study provides important information on neuronal growth‐cone durotaxis, demonstrating neurite orientation in response to the presence of a stiffness gradient, and revealing previously unreported sensitivity to the combination of gradient slope and absolute stiffness. Neurites grew toward stiffer or softer substrate regions, depending on the gradient/absolute stiffness combination, highlighting how both cues combine to control orientation. This work presents a promising strategy for future neural tissue engineering approaches that harness mechanical features of biomaterials to support and guide neuronal regeneration.The authors declare no conflict of interest.


While stored, more short-range double helices, long-range crystallites, and nanoscale orders emerged for the microwave-cooked starch than for its conventionally cooked counterpart. For instance, after storage for 24 h, the microwave-cooked starch contained 46.8% of double helices, while its conventionally cooked counterpart possessed 34.3% of double helices. This could be related to the fact that the microwave field caused high-frequency movements of polar groups such as hydroxyls, which strengthened the interactions between starch chains and water molecules and eventually their assembly into double helices, crystallites, and nanoscale orders. This work provides further insights into the chain reassembly of microwave-cooked starch undergoing storage, which is closely related to the quality attributes of starch-based products.The chain reorganization of cooked starch during storage plays an important role in the performance of starchy products such as rice foods. Here, different analytical techniques  were used to reveal how microwave cooking influences the chain assembly of cooked  This polymer contains two types of glucan polymers, including amylose (molar mass: ~108 g/mol) . The str8 g/mol) ,4. Befor8 g/mol) ,6,7,8. T8 g/mol) ,10, and 8 g/mol) ,12,13,148 g/mol) ,16. Furt8 g/mol) ,18,19. Tindica rice starch undergoing storage. It is known that indica rice accounts for more than 80% of rice cultivation worldwide and is an important staple food for more than half of the world population [There have been reports on the retrogradation of starch cooked using different methods, such as extrusion ,21,22, apulation ,39.indica rice was used as the material for starch isolation. The starch slurries were prepared and cooked using microwave heating and conventional heating. Then, the cooked starch samples were stored for different time periods . Different analytical methods were used to inspect the multi-scale structural changes of the starch during the course of storage. The present data are of value for the quality regulation of rice foods subjected to microwave treatment.Here, Indica rice was obtained from Xiangyang Saiya Rice Co., Ltd. . The starch was extracted according to our previous method [indica or low-amylose rice grains [s method . The amye grains ,41. The e grains . All othw/v). The slurry was heated in a microwave oven  at 8 W/g for 3 min. Each of the gelatinized samples was cooled and stored at 4 °C for 24 or 48 h and dried. The freshly cooked starch was freeze dried. The dried samples were crushed and passed through an 80-mesh sieve. The samples were denoted using codes such as “MC-24”, in which MC represents microwave cooking and 24 indicates the storage time. For control studies, the starch slurries (30% w/v) were heated in boiling water for 30 min to acquire conventionally cooked starches, which were dried and crushed according to the above method. The conventionally cooked starches were denoted using codes such as “C-24”.The starch was placed in distilled water to obtain a starch slurry with a starch concentration of 30% . The starch samples were placed on conductive tapes and coated with a thin gold film before observations at an acceleration voltage of 15 kV.q range between 0.015 and 0.5 Å−1 from the 2D scattering patterns using the RAW software. The scattering vector q was equal to 4πsinθ/λ .The synchrotron SAXS measurements were performed on the BL19U2 BioSAXS beamline at the Shanghai Synchrotron Radiation Facility . All the samples were dispersed into distilled water until the moisture reached 80% and equilibrated at room temperature. Then, the samples were filled into 2 mm-thick sample cells, which were covered with Kapton tape. The 2D scattering pattern for each starch was recorded at a wavelength of 1.03 Å using a Pilatus 1 M detector . The 1D scattering curves were obtained in a θ angle range of 5° to 40° at 40 kV and 40 mA, with a Cu Kα X-ray source. The relative degree of crystallinity was estimated using the method by Lopez-Rubio, Flanagan, Gilbert, and Gidley [The X-ray diffractograms of the samples were obtained using an X-ray diffractometer  at a scanning speed of 0.5 °/s and a step size of 0.02°. The starch powders were tested through a 2d Gidley .−1. Each spectrum was recorded at a resolution of 4 cm−1 for 32 scans against the air as the background. The spectra in the range of 1200 to 800 cm−1 were smoothed, baseline corrected, and deconvoluted using the Omnic 8.2 software. The ratio of the peak intensity at 995 cm−1 to that at 1022 cm−1 (R995/1022) was used to indicate the relative amount of starch short-range orders.The FTIR spectra of the samples were obtained on a Nicolet iS50 infrared spectrometer  equipped with an ATR accessory from 4000 to 400 cm1CP/MAS 3C NMR spectra were collected at a 13C frequency of 100.613 MHz on an Advance AV spectrometer , operating at 295.6 K and using a 4 mm broadband double-resonance MAS probe. Approximately 400 mg of the starch powder was placed into a 4 mm-diameter magic-angle spinning sample rotor with a tight push-fitting cap. At least 3000 scans were accumulated for each spectrum with a recycle delay of 2 s. The relative proportions of starch single helices, double helices, and amorphous material were calculated using the PeakFit software  [The solid-state IL, USA) .p < 0.05 was considered to be significant.The data were expressed as the means ± standard deviations. The statistical analysis was performed using a one-way ANOVA via the 20.0 IBM SPSS software . A statistical difference at indica rice starch at the different storage time periods of 0, 24, and 48 h, as shown in −1) indicated the existence of nanoscale (approximately 15–20 nm) starch molecular orders that were randomly distributed in amorphous matrices  [The SAXS patterns were used to investigate the nanostructure of cooked uctures) . The mic−1 (about 1.6 nm in size) was observed for the two starches, ascribed to the presence of a B-type crystalline structure [Storage for 24 h strengthened the shoulder peak of conventionally cooked starch (C-24), but the microwave-cooked starch (MC-24) showed a more intense shoulder peak than C-24. This result again confirms the presence of more nanoscale molecular orders for the microwave-treated starch with storage, and these orders aligned with amorphous regions to construct the nonperiodic structure . The higtructure ,54 , which are usually examined by XRD . Figure allinity . This imallinity . Compare-1 for the starch samples are shown in −1 is associated with the relative content of amorphous starch materials, and the band at ca. 995 cm−1 reflects the content of short-range orders such as starch helices [R995/1022) of the band at 995 cm−1 to that at 1022 cm−1 can be used to indicate the content of short-range orders, and the results are presented in R995/1022 close to that of the starch subjected to microwave treatment (MC-0). While storage for 24 h resulted in a comparable R995/1022 for the two types of cooked starches, a longer storage time did not further change the R995/1022 value. It has been found that the short-range order absorbance ratio of banana starch reaches a plateau after storage for 11 h [Two adjacent branches of amylopectin could form double helices (short-range ordered structures) . The ATR helices ,58. The for 11 h . Conside13C CP/MAS NMR has been applied to estimate the relative proportions of single- and double-helical components of starch [13C CP/MAS NMR profiles of the starches and their ordered sub-profiles are presented in f starch ,60. The The molecular chains of cooked starches can reassemble into ordered structures during storage, largely affecting the properties of starch matrices such as hardness and digestibility. This chain reassembly event involves several key steps: (i) amorphous chains are organized into single helices (via hydrophobic force) and double helices (via hydrogen bonds); (ii) the single helices aggregate into V-type crystallites, and the double helices align with water molecules via hydrogen bonds to construct monoclinic crystal cells  and hexagonal crystal cells ; (iii) the helical and crystalline components further aggregate to form nanoscale ordered regions, and these regions randomly distribute in amorphous regions ,13,40. TThe theory here and the above results could enable an understanding of how microwave cooking followed by storage induces more nanoscale and short-range orders in starch. Specifically, the two cooking methods sufficiently disrupted the semicrystalline architecture of native starch, as indicated by the residues with similar amounts of short-range scale orders in cooked starches. Unlike conventional (conductive) cooking, the electromagnetic effects of microwave cooking could induce high-frequency movements of polar species, such as hydroxyl groups of starch chains and water molecules. This fact probably increased the opportunities for starch chain–water interactions. Consequently, microwave cooking followed by storage not only substantially enhanced the formation of short-range orders (chain self-assembly into helices) during storage but also facilitated the construction of long-range crystallites (containing helices with water molecules) and then nanoscale orders (aggregation of short- and long-range orders). Furthermore, the greater number of long-range scale and nanoscale orders in microwave-cooked starch could serve as seeds to promote the formation of ordered structures.This work confirms that microwave-cooked starch following storage possessed more short-range, long-range, and nanoscale ordered structures, relative to the conventionally cooked starch. While the two cooking manners reserved a similar number of short-range helices, the electromagnetic effects of microwave heating likely induced the high-frequency movements of polar groups (such as hydroxyls) and thus enhanced the interactions between starch chains and water molecules. This was preferable for the chain alignment into short-range double helices, long-range crystallites (aggregated from helices and water molecules), and nanoscale orders . Consistently, storage for 24 h led to more double helices (46.8%), B+V crystallites, and nanoscale orders for the microwave-cooked starch than for its conventionally treated counterpart. A further increase in the storage time slightly affected the ordered structural features on different scales. This work enables a better understanding of the chain reassembly of microwave-cooked starch during storage, which is of value for the design of starch-based products (such as ingredients) with tailored qualities such as digestibility.


However, overexpression of the auxin-inactivating iaaL gene in rootstocks delayed graft healing. We observed increased endogenous auxin levels and auxin-responsive DR5::GUS expression in scions of WT/iaaM grafts compared with those found in WT/WT grafts, which suggested that auxin is transported upward from rootstock to scion tissues. A transcriptome analysis showed that auxin enhanced graft union formation through increases in the expression of genes involved in graft healing in both rootstock and scion tissues. We also observed that the ethylene biosynthetic gene ACS1 and the ethylene-responsive gene ERF5 were upregulated in both scions and rootstocks of the WT/iaaM grafts. Furthermore, exogenous applications of the ethylene precursor ACC to the junction of WT/WT grafts promoted graft union formation, whereas application of the ethylene biosynthesis inhibitor AVG delayed graft healing in WT/WT grafts, and the observed delay was less pronounced in the WT/iaaM grafts. These results demonstrated that elevated auxin levels in the iaaM rootstock in combination with the increased auxin levels in scions caused by upward transport/diffusion enhanced graft union formation and that ethylene was partially responsible for the effects of auxin on grafting. Our findings showed that grafting success can be enhanced by increasing the auxin levels in rootstocks using transgenic or gene-editing techniques.Grafting is a highly useful technique, and its success largely depends on graft union formation. In this study, we found that root-specific expression of the auxin biosynthetic gene  In the horticultural and silvicultural industries, grafting is widely used to increase the yield, enhance biotic and abiotic stress resistance, and modify the scion architecture3. Although grafting has been used for millennia in agriculture, the mechanisms underlying the graft healing process are largely unclear. Previous studies have described the initial cell proliferation that occurs at the beginning of graft union formation, and this step is followed by the formation of a mass of pluripotent, undifferentiated cells known as calli. The callus then differentiates into vascular tissues to reconnect the phloem and xylem across the graft junction, which leads to a successful graft union5.Grafting is an ancient but essential technique used worldwide in the agricultural industry that involves combining plants of the same or different species to continue their growth and development5. Exogenous auxin application can lead to successful graft formation during tissue culture6. Conversely, hypocotyl graft unions are inhibited in response to treatment with an auxin transport inhibitor7. Indeed, many Arabidopsis thaliana mutants with impaired auxin perception or auxin responses show delayed phloem reconnection during hypocotyl grafting4.Various plant hormones are involved in the graft healing process. Auxin accumulates above the graft junction and is depleted at the bottom of the junction9. The auxin response factors ARF6 and ARF8 are essential for cell proliferation during tissue reunion. The arf6 arf8 double mutant shows strongly inhibited cellular proliferation during tissue reunion in incised Arabidopsis inflorescence stems compared with that obtained with the wild type10. Another important gene for grafting, ABERRANT LATERAL ROOT FORMATION 4 (ALF4), acts downstream of auxin signaling and is specifically required in the rootstock for normal phloem reconnection during graft union formation4. Moreover, auxin induces the expression of the xyloglucan endo-transglucosylase/hydrolase genes XTH20 and XTH19, which encode proteins involved in cell proliferation during the tissue reunion process, in the distal parts of incised stems12.Transcriptome analyses have uncovered a series of auxin response genes involved in the grafting process5. Ethylene is involved in the wounding response and promotes cell expansion14, and the exogenous application of ethylene promotes callus formation to result in increased cell proliferation15. The ethylene signaling-defective Arabidopsis mutant ein2 shows incomplete healing during tissue reunion, and incised inflorescence stems of this mutant exhibit inhibited expression of the transcription factor gene ANAC071, which is needed for the division of pith cells during reunion. The expression of ANAC071 is also induced by auxin during this process11. Moreover, the application of the ethylene biosynthesis inhibitor AVG (2-aminoethoxyvinylglycine) to auxin -supplemented medium inhibits callus formation at the base of the shoot15. These findings indicate that ethylene and auxin interact during graft union formation, but further studies are needed to elucidate the detailed mechanism.Auxin also interacts with other hormones during graft union formationiaaM (one tryptophan-2-monooxygenase gene from Agrobacterium tumefaciens) is expressed predominantly in roots, as the rootstock for grafting to a wild-type (WT) scion to study the role of endogenous auxin in the graft healing process. This Agrobacterium gene iaaM, when inserted into plants, can convert tryptophan to indole-3-acetamide, and this product is then converted to the active phytohormone indole-3-acetic acid (IAA) by endogenous hydrolases16. The overexpression of iaaM in rootstock led to an increase in the auxin levels and upregulated the expression of genes associated with graft union formation in WT scions. Our findings indicate that ethylene promotes graft union formation and is involved in rapid auxin-mediated callus formation during the graft healing process.Here, we used auxin-overproducing transgenic tobacco, in which iaaM gene driven by the root-predominant promoter SbUGT, which is a flavonoid glycosyltransferase gene from Scutellaria barbata17, is expressed predominantly in the roots of tobacco  plants as the rootstock for grafting studies. One week after grafting, we observed complete graft healing with no gaps in the WT/iaaM grafts but only partially healed graft unions with clearly deeper gaps in the WT/WT grafts  gene driven by the shoot-specific SAUR promoter19. The iaaL gene encodes an indole-3-acetic acid-lysine synthetase that catalyzes the conversion of free auxin into inactive indole-3-acetyl-L-lysine20. Here, we used SAUR::iaaL (iaaL) plants as the rootstock for WT scions. The reduced endogenous auxin levels caused by iaaL expression in rootstocks exerted severe effects on graft healing. One week after grafting, we observed larger gaps in the unions of the WT/iaaL grafts than in those of the WT/WT grafts. Among the graft combinations , the WT/iaaM grafts exhibited the best healing , were higher at both 24 and 96 h after grafting than in the rootstock of the WT/WT grafts , which is critical for phloem reconnection8, showed 2.5-fold higher expression in the iaaM rootstock than in the WT rootstock  to the WT/WT graft junctions. Three weeks after grafting, scion growth was enhanced by ACC application but reduced by AVG application compared with the levels found with the control treatment  cohesion of the scion and rootstock; (ii) proliferation of callus cells at the graft interface; and (iii) vascular redifferentiation across the graft interfaceArabidopsis by exogenous application or decapitation11. Auxin is also involved in union formation in Arabidopsis hypocotyl grafting, which is not completely the same as wound healing7. Most studies have focused on auxin accumulation above the graft junction during grafting, whereas Melnyk et al4. found that auxin-responsive genes involved in vascular reconnection, such as ALF4 and transport inhibitor response protein 1 (TIR1), are needed below the graft junction. Here, we showed that endogenous auxin in rootstock also plays an important role in grafting, as demonstrated with SbUGT::iaaM or SAUR::iaaL rootstock. Our results revealed that the auxin-mediated improvement of graft union formation is largely due to enhanced expression of genes important for callus and vascular cell development/reconnection. However, in Arabidopsis hypocotyl grafting, decreases in the endogenous auxin levels associated with p35S::iaaL (WT/p35S::iaaL) do not affect the phloem connection at 4 days after grafting4. The inconsistency of the results might be due to differences in organs or species.The positive role of auxin in the partial incision healing process has been well demonstrated in the model plant 30. Incompatibility between scions and rootstocks restricts the broad use of the grafting technique among different plant varieties18. In a compatible graft, the callus fills up the spaces between the scion-and-rootstock junction, which holds the two tissues together tightly, whereas a necrotic layer is always observed in incompatible grafts31. Auxin levels are correlated with graft compatibility32. Indeed, in a study of the homograft Litchi chinensis cv. ‘Jingganghongnuo’ (compatible graft) and the heterograft ‘Jing ganghongnuo’/‘zhuangyuanhong’ (incompatible graft), higher IAA levels were observed in compatible graft unions31. In the current study, we also found that increasing the endogenous auxin levels in the rootstock can improve the rate of successful grafting. Therefore, the overexpression of iaaM in rootstock has great potential for improving the rates of successful grafting with incompatible grafting partners.Graft compatibility is needed for successful grafting34. The ethylene-deficient Arabidopsis mutant ein2 exhibits delayed tissue reunion compared with the WT in cut inflorescence stems11. Moreover, ethylene promotes callus formation in cultured cotton ovules by stimulating both cell division and cell expansion35. In the current study, we applied the exogenous ethylene biosynthesis precursor ACC and the ethylene biosynthesis inhibitor AVG to the WT/WT graft junctions. Our results demonstrated that ethylene plays a positive role in graft union formation. However, the ethylene-overproducing Arabidopsis mutant ctr1-1 adversely affects the phloem connection in hypocotyl grafts4. A probable reason for this contradiction is the double-edged sword role of ethylene. Transgenic plants with elevated auxin levels also exhibited increased ethylene levels. An analysis of transcriptome data revealed that the expression of genes related to ethylene biosynthesis and responsive signaling pathways, such as 1-aminocyclopropane-1-carboxylate oxidase homolog 1 (Nitab4.5_0002916g0060.1), ethylene-responsive transcription factor (Nitab4.5_0007739g0010.1), and probable WRKY transcription factor 33 (Nitab4.5_0001226g0020.1), was markedly higher in the WT/iaaM grafts than in the WT/WT grafts. Additionally, AVG treatment partially delayed graft union formation in the WT/iaaM grafts, and callus formation induced by NAA treatment was inhibited by the application of AVG. These results suggest that ethylene is involved in the action of auxin during grafting and that the promoting effect of ethylene on grafting is likely due to its positive effect on callus formation in scion-and-rootstock junctions.Ethylene functions as a wounding response signalArabidopsis9, but the detailed mechanism has not been well characterized. Because auxin can be transported short distances in a cell-to-cell manner through protoplasts, this phytohormone is thought to be transported across the graft junction prior to vascular reconnection9. Our transcriptome analysis identified a series of genes that are differentially expressed between the WT/iaaM and WT/WT scions prior to the formation of graft unions (24 h after grafting); although a number of genes, such as Wound Induced Protein 136, Lateral Organ Boundaries Domain 16 (LBD 16), LBD17, LBD1837, Histone H48, NAC096, NAC0717, and HCA28 have been identified, the exact mechanism responsible for graft union formation has not been illuminated. These genes function in multiple processes, including wounding responses, cell division, cell proliferation, vascular formation, and hormone responses. Among these processes, wounding responses are involved in graft union formation. NRT3.1 (Nitab4.5_0003741g0010.1) was upregulated by 18.23-fold in the WT/iaaM compared with the WT/WT scions. This gene encodes a high-affinity nitrate transporter involved in jasmonic acid-independent wound signal transduction, which suggests its potential involvement in wounding responses during graft union formation. Nitrate can also function as a signaling molecule38. Although NRT3.1 was upregulated in the scions of the WT/iaaM grafts compared with those of the WT/WT grafts, whether it participates directly or indirectly in auxin signaling pathways requires further study. By employing plants harboring the auxin reporter gene DR5::GUS as the scion and measuring the endogenous IAA levels, we demonstrated that increased auxin levels in the rootstock can lead to high auxin levels in the scions 24 h after grafting, i.e., before vascular reconnection, and this effect might be due to passive diffusion.Numerous studies have suggested that communication between the scion and rootstock promotes graft healing. For instance, a signal exchange process between scion and rootstock cells has been observed 24 h after hypocotyl grafting in iaaM rootstocks initially showed enhanced root biomass, more vigorous scion growth, and no lateral bud release compared with the WT rootstocks, the application of SbUGT::iaaM expression as a practical technology is not the best option due to an observed reduction in root growth39. Although we revealed the mechanism of graft union formation, we used iaaM plants to demonstrate that a high auxin content in the rootstock can result in a high auxin content in the scion regardless of vascular reconnection, and this effect is likely due to the upward transport or passive diffusion of auxin and accelerates callus proliferation and graft union formation. Our results suggest that auxin promotes graft union formation through interactions with ethylene, and these findings uncover great potential for the action of auxin during grafting.Although the iaaM fusion gene can enhance grafting success through a series of physiological and molecular events in both rootstock and scion tissues. At the physiological and morphological levels, increased auxin levels enhance root initiation and inhibit lateral bud release in rootstocks, and these higher levels also promote more vigorous scion growth at early stages after grafting. At the biochemical level, elevated auxin concentrations in rootstock tissue also result in a high auxin content in scion tissues due to the upward transport or passive diffusion of auxin. Ethylene plays a positive role in callus formation and vascular tissue development/reconnection, and elevated auxin levels in rootstock tissue also induce more ethylene production in both rootstock and scion tissues. At the molecular level, we observed that the expression of a series of genes involved in callus formation, vascular tissue development, and vascular tissue connecting/graft union formation between rootstock and scion tissues, including Wound induced protein 1 (WIND1), Lateral Organ Boundaries Domain (LBD) 16, LBD17, LBD18, Histone H4, cyclin genes, such as CYCA1-1,CYCA2-2, CYCB2-3, CYCB2-4 and CYCD3-1, the NAC domain-containing proteins NAC096 and NAC071, High Cambial Activity 2 (HCA2), Expansin-A15, and Expansin-like B1, are enhanced in both rootstock and scion tissues37 when iaaM transgenic rootstock is used.Based on the data presented in this manuscript, we propose a working model for the mechanism underlying the effects of auxin-enriched rootstock on grafting Fig. . We propSbUGT::iaaM tobacco 39 and SAUR::iaaL tobacco (iaaL tobacco) were propagated on Murashige and Skoog medium  from PhytoTechnology Laboratories (USA). The plants were grown in a tissue culture room at 25 ± 2 °C with a 16-h photoperiod and a light intensity of 250 μmol quanta/m2/s. After two months of growth in MS medium, all iaaM tobacco and WT plants were used for grafting.iaaM, and iaaL stems were used as rootstocks. WT apical shoots were used as scions. A scion with a wedge shape cut at the basal end was inserted into a rootstock with a longitudinal cut at the apical end . The degradation and contamination of RNA were monitored using 1% agarose gels, and the RNA purity was checked using a NanoPhotometer® spectrophotometer . The RNA integrity and quantitation were assessed using an RNA Nano 6000 Assay Kit and the Bioanalyzer 2100 system . One microgram of RNA from each sample was used for library preparation. Sequencing libraries were generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations. The library quality was assessed using the Agilent Bioanalyzer 2100 system.Scion and rootstock tissues of the WT/WT or WT/Nicotiana tabacum genome using TopHat v2.0.12. The gene expression levels were determined based on the fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) values. Differential expression analysis comparing two treatments was performed using the DEGSeq R package (1.20.0). A corrected P-value of 0.005 and a log2(fold change) value of 1 were used as thresholds to identify differentially expressed genes.The raw data (raw reads) were processed by removing adapter sequences. Clean reads were obtained by removing reads containing adapter and poly-N sequences and low-quality reads with low Q-values. The clean reads were mapped to the DR5::GUS construct40 was introduced into Agrobacterium tumefaciens strain EHA105, and the resulting bacteria were used to transform Nicotiana tabacum cv. Xanthi. Tobacco leaf disc transformation was performed as described previously41. Genomic DNA was extracted from the leaves of putative transgenic plants using a Plant Genomic DNA Extraction Kit . The DNA was purified and used as a template for PCR42. The primer pair GUS-F (5′-TCTTCGACCTCAATGGCG-3′) and GUS-R (5′-ACGAATGACTTTTCCGAGG-3′) was used to amplify a 443-bp fragment from the GUS reporter gene within the T-DNA region of the Ti plasmid. PCR was performed using EmeraldAmp® GT PCR master mix . The reaction solution (20 µL) included 10 µL of 2× PCR mix, 0.5 µL of the forward primer, 0.5 µL of the reverse primer, 500 ng of the template DNA, and ddH2O. The PCR cycling conditions were as follows: 98 °C for 5 min; 35 cycles of 98 °C for 10 seconds, 60–65 °C for 30 seconds, and 72 °C for 1 minute/kb; and a final extension at 72 °C for 10 min.The DR5::GUS shoots were placed in MS medium for one day to induce the accumulation of endogenous auxin in the basal end. Approximately 0.5 cm of the shoot basal end was subsequently cut off, and the rest of the shoot was used as a scion for grafting onto WT or iaaM rootstock. Twenty-four hours after grafting, the scions were cut longitudinally in the middle of the stem and incubated in X-Gluc solution at 37 °C overnight for histochemical GUS staining. The X-Gluc solution consisted of 100 mM potassium phosphate buffer, pH 7.0, 10 mM Na2EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1% Triton X-100, and 1 g/L X-Gluc (5-bromo-4-chloro-3-indolyl-b-d-glucuronic acid). After staining, chlorophyll and other pigments were removed from the samples using successive concentrations of ethanol  prior to visual inspection and imaging. SbUGT::GUS and 35 S::GUS transgenic tobacco seeds43 were germinated in Petri dishes. After one week of growth, the seedlings were subjected to GUS staining as described above.Prior to grafting, iaaM grafts were harvested at 0, 24, and 96 h after grafting. Sample collection was performed as described for RNAseq analysis. The samples were weighed and frozen in liquid nitrogen. The IAA and ACC measurements were performed by HPLC-ESI-MSn (high-performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry) at Beijing Forestry University45. The mean hormone contents of the WT/WT and WT/iaaM grafts were compared using two-tailed Student’s t-test with pooled variance.The scions of the WT/WT or WT/P = 0.05) was used to calculate the differences between treatments.Exogenous treatments were performed using the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (5 mM) and the ethylene biosynthesis inhibitor 2-aminoethoxyvinylglycine (AVG) (0.5 mM). Scions and rootstocks were treated with ACC or AVG prior to grafting, and treatment with water was included as the control. The cut scions and rootstocks were dipped into the treatment fluid for 3 seconds and then immediately grafted. Three weeks after grafting, the scion of each graft was harvested and dried at 105 °C for 30 min before weighing. The data are presented as the means from six replicates. Analysis of variance (ANOVA) was performed with IBM SPSS 19.0 . Fisher’s protected least significant difference (LSD) test , incubated overnight in fresh 0.05 M PIPES at 4 °C and rinsed for 40 min the next morning. Secondary fixation was conducted in 1% osmium tetroxide, 0.8% potassium ferricyanide, 1.5 mM MgCl2, and 1.5 mM CaCl2 in 0.05 M PIPES, pH 6.9, for 40 min at room temperature, and the samples were then incubated at 4 °C for 40 min. Prior to dehydration, the tissue was subjected to three 15-min rinses in cold Milli-Q distilled water . The tissue was dehydrated twice through a graded ethanol series  and cleared twice with propylene oxide for 10 min each time. Spurr’s resin ethanol) was freshly prepared. The tissue was infiltrated in a 1:3 mixture of resin:propylene oxide for 1 h, a 1:1 mixture of resin:propylene oxide mixture for 3 h, and a 3:1 mixture of resin:propylene oxide overnight. The following day, the tissue was infiltrated in 100% Spurr’s resin for 7 h and incubated in fresh resin overnight. The next day, the tissue was infiltrated in 100% Spurr’s resin for 6 h. The samples were then placed in flat round molds  and polymerized in an oven  under a vacuum at 60 °C for 48 h.Tissue samples were incubated in a solution of 2.5% glutaraldehyde, 2.0% paraformaldehyde, 1.5 mM MgClSemithin sections (~1 µm) were cut with a Histo 45° Diatome™ diamond knife on a Leica Ultracut UCT microtome and collected on drops of 10% acetone on Superfrost® Plus microscope slides (Fisher Scientific). The sections were dried by placing the slide drip-side down over a small open Petri dish (previously heated) for evaporation on a hotplate, stained at 70 °C with a solution of 1:1 methylene blue:azure blue II, and placed on a 30-8010 AB slide warmer (Buehler Ltd.) for 15 seconds at 90 °C. The sections were examined under an Olympus light microscope. All steps were conducted at room temperature in glass shell vials with plugs (Fisher Scientific) on a Pelco R2 rotary mixer  at setting 1 to facilitate penetration of the chemicals unless otherwise indicated.P = 0.05) was used to calculate the differences between treatments.Tobacco internode stems (~2 mm) were placed on MS medium supplemented with 5 µM NAA, 10 µM AVG, or 10 µM ACC for callus induction. After three weeks, the callus was carefully removed with a scalpel and weighed. Eight to ten stems were grouped together to obtain one replicate. The data are presented as the means from three replicates. ANOVA was performed with IBM SPSS 19.0 software . Fisher’s protected LSD test (Fig. S1 and S2Table S1


Previous studies have argued that people tend to isolate themselves from negative information. This tendency is modulated by the individual's role in social interaction, that is, as an initiative actor  or a passive recipient . Depressed patients tend to focus on negative aspects of themselves and cope with situations passively. It is still an open question how the actor/recipient role affects the behavioral and neural responses to self in depression.The present study adopted functional magnetic resonance imaging (fMRI) technology to investigate behavioral and neural responses to self (as an actor/recipient) in depressed patients and the matched healthy controls when attributing negative events.Compared with healthy controls, depressed patients showed more self‐attribution for negative events. Depressed patients showed increased brain activity in the dorsal medial prefrontal cortex (dmPFC) subsystem of the default mode network (DMN) when they played recipient role in self‐related negative events. Activity of the dmPFC subsystem was negatively correlated with depressed patients’ self‐attribution for negative events in recipient condition. While decreased brain activity in the medial temporal lobe (MTL) subsystem was observed in depressed patients when they played the actor or recipient role in self‐related negative events. Activity of the MTL subsystem was negatively correlated with depressed patients’ reaction time when they played recipient role in selfrelated negative events.These results implicated that depressed patients manifested the negative self‐view. Actor/recipient role affected their activation patterns in the DMN which were different from the healthy controls. The correlation between the abnormal brain activations of the DMN and the behavioral performances might manifest more easily when depressed patients played recipient role in negative events. Depressed patients showed more self‐attribution for negative events than healthy controls.Depressed patients showed increased brain activity in the dmPFC subsystem and decreased brain activity in the MTL subsystem of the default mode network (DMN).Brian activity of the DMN was negatively correlated with depressed patients' level of self‐attribution or reaction time when “self” was the passive recipient of the negative events. The exclusion criteria for the depressed patients included co‐morbid psychiatric illness, substance abuse within 6 months of screening, prior electroconvulsive therapy treatments, and a history or current symptoms of psychosis or mania. The severity of affective symptoms was assessed 1−7 days prior to scanning with the 21‐item Beck Depression Inventory  or recipient role , and the self was the target of evaluation . For other‐relevant events , both the actor  and recipient  would become the targets of evaluation separately . Thus, these stimuli can be categorized into four conditions: actor‐self (AS), actor‐other (AO), recipient‐self (RS), and recipient‐other (RO).Participants were asked to complete 80 trials in the scanner. In each trial, participants were presented simultaneously with a one‐sentence interpersonal event, an evaluation question, and a 4‐point scale . They were asked to imagine the event happening to them or to others and make an evaluation within 8 s by pressing the corresponding button. Each trial was jittered with inter‐stimuli intervals from 200 to 1000 ms, during which a black fixation cross was presented against a white background. All trials were randomly intermixed and equally divided across two runs.2.3o) line. A high‐resolution fast‐spoiled gradient recall T1‐weighted image was also acquired from each participant .All images were collected on a 3T GE Discovery MR750 scanner at the Qiqihar Mental Health Center. Functional images were acquired with an EP∖GR sequence oriented to the AC‐PC  using the normalization parameters from T1 image estimated during unified segmentation, and then spatially smoothed with a Gaussian kernel of 8‐mm full‐width at half‐maximum. A high‐pass filter with a cutoff period of 128 s was applied.Data analyses were conducted with SPM8 . Preprocessing included discarding the first five functional images to allow for scanner equilibrium effects. The remaining 450 functional images were corrected for the delay in slice acquisition and were spatially realigned to the first image. The individual T1‐weighted, 3D structural image was co‐registered to the mean EPI image generated after realignment. The co‐registered structural image was then segmented into gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) using a unified segmentation algorithm. The functional images after slice timing and realignment procedures were spatially normalized into the Montreal Neurological Institute (MNI) space  were estimated since this number offers a good trade‐off between preserving a massive amount of information in the data and reducing the size of the datasets. Third, group spatial ICA was conducted using the Infomax algorithm, which is one of the commonly used ICA algorithms.2.4.3The GLM design matrix included the four experimental conditions  modeling the attribution task, a fixation condition modeling the baseline, six regressors modeling movement‐related variance, and one regressor modeling the overall mean. For the four experimental conditions of interest, we chose the onset of the stimulus as the onset time point and the RT from the stimulus onset to button press as the duration (epoch with variable time length).2.4.4r2 < 0.05, r2 < 0.02 for WM, and r2 > 0.05 for GM . The components that passed the three criteria were regarded as valid for further analysis.There were three steps to select the components of interest. First, each component spatial map was correlated with prior probabilistic maps of gray matter (GM), white matter (WM), and CSF within a standardized brain space offered by the MNI templates in SPM8 .The remaining components in the selection were then subjected to a one‐sample 2.4.6Finally, in order to determine how these brain activities were regulated by the task, we calculated separately the correlations among beta weights, participants’ attribution ratings, and the corresponding reaction times in the experimental conditions.33.13.1.1F  = 30.78, p < .001], and a significant interaction between Group and Target . There were no other main effects or significant interactions . Further simple effect analysis revealed that, for self‐related negative events, depressed patients’ attribution ratings were higher than those of healthy controls . In contrast, for other‐related negative events, there was no difference in attribution rating between the two groups   × 2 (Target: self vs. other) × 2 (Role: actor vs. recipient) repeated measures ANOVA revealed that participants’ attribution ratings were characterized by a main effect of Target , and a significant interaction between Group and Role . There were no other main effects or significant interactions .A 2 (Group: DP vs. HC) × 2 (Target: self vs. other) × 2 (Role: actor vs. recipient) repeated measures ANOVA revealed that participants’ reaction times (RTs)  = 21.10, p < .001, while there was no significant difference between the two conditions in depressed patients, F  = 0.17, p = .68. Additionally, we found that depressed patients’ responding were marginally slower than healthy controls in recipient condition  but not in actor condition .For the interaction between Group and Role, further simple effect analysis revealed that healthy controls responded slower in actor condition than in recipient condition, 3.23.2.1p < .05, 164 contiguous voxels, with an underlying voxel level of p < .01 uncorrected), the remaining four components were identified in the fMRI results . There was no other significant difference between the DP and HC groups under other conditions, all t < 1.98, all p > .06  but not in the HC group .To further identify how these group differences in brain activities in each component were regulated by the corresponding experimental condition in the task, correlation analyses were conducted. The results showed that the beta weights of the dmPFC subsystem  in the RS condition were negatively correlated with individuals’ attribution ratings in the DP group  but not in the HC group . There was no other significant correlation between the beta weights of these components and behavioral performance in either the HC or the DP group in the four experimental conditions .The beta weights of the MTL subsystem  in the RS condition negatively correlated with individuals’ RT in the DP group . Moreover, the greater the activity in the dmPFC subsystem of a depressed patient, the less self‐attribution for negative events was observed in the RS condition. Additionally, we found less activity in the MTL subsystem in depressed patients than in healthy controls when they played the actor or recipient role in self‐related negative events. But depressed patients only showed a negative correlation between MTL activity and reaction time in RS condition.A central feature of depressed patients is having elevated feelings of self‐blame and paying more attention to negative aspects of themselves (Korn et al., In contrast, we found that the MTL subsystem of the DMN was less activated in depressed patients than in healthy controls. Previous studies have argued that these regions are associated with autobiographical memory and information retrieval (Viard et al., Consistent with the previous studies on the self‐processing of depression using the attribution rating task, we confirmed that depressed patients manifested negative self‐view and different activation patterns in the DMN from the healthy controls. Importantly, we distinguished the role played by the self in interpersonal events, that is, an actor or a recipient, in the present study. We found preliminarily that actor/recipient role affected depressed patients’ activations of the DMN. The correlation between the abnormal brain activations of the DMN and the behavioral performances might manifest more easily when depressed patients played recipient role in negative events. However, there were limitations to the present study that should be acknowledged. All but four of the depressed patients were receiving antidepressant medication at the time of the study, which meant that the results might be confounded by medication status. Additionally, the sample size of the present study may have been too small to provide strong evidence to illustrate the neural mechanism of how the actor/recipient role affects one's emotional self‐processing. Therefore, caution should be applied when interpreting these findings, and further research is needed to better understand the effect of the actor/recipient role on self‐cognition in depressive disorders.5To the best of our knowledge, this was the first study that explored the underlying neural correlates of how the actor/recipient role affects negative self‐processing in depressive disorders. Compared with healthy controls, depressed patients exhibited greater activity in the dmPFC subsystem and less activity in the MTL subsystem of the DMN. Especially when depressed patients were passively subjected to negative interpersonal events, brain activity in the dmPFC subsystems was negatively correlated with self‐attribution for negative events. These results suggested that increased brain activity in the dmPFC subsystem and decreased brain activity in the MTL subsystem of the DMN could be a potential biomarker for depression. The results also highlighted the importance of exploring the effect of the actor or recipient role on self‐processing in social situations.The authors report no conflict of interest.https://publons.com/publon/10.1002/brb3.2477The peer review history for this article is available at


SPT is linked to many diseases including fungal infection, making it a potential therapeutic target. Thus, a logical docking-based virtual screening method was used to screen selective SPT inhibitor against fungi, not human. We used myriocin-similarity database to identify compounds with good binding with fungal SPT and poor binding with homology human SPT model. Preliminary bio-assay led to the discovery of a promising inhibitor WXP-003, which displayed good inhibitory activity against diversity fungi strains with MIC ranging from 0.78 to 12.5 μg/mL. WXP-003 could significantly reduce sphingolipids content in fungi and no effect on mouse fibroblast cell line L929. Molecular dynamics simulation depicted that SPT/WXP-003 complex formed the favoured interactions. Taken together, discovery of WXP-003 provided valuable guide for the development of novel anti-fungal agents.Serine palmitoyltransferase (SPT) plays the key role on catalysing the formation of 3-ketodihydrosphingosine, which is the first step of the  Based on myriocin is a well-known SPT inhibitor, we hypothesised myriocin-analogues might have higher chances on good binding with SPT2.2.1.https://www.ncbi.nlm.nih.gov/protein/). BLAST search(BLASTP) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was employed to search the template proteins for building human SPT, the search database was set to protein data bank (PDB), and the other parameters used defaults. After sequences search, four proteins structures were chosen, namely, 2X8U (PDB ID)Align Sequence to Templates module of Discovery Studio 3.5 (DS3.5) was employed to align the human SPT sequence and the template protein sequences. Second, the Build Homology Models module in DS3.5 was used to construct the target protein with the above sequence alignment, and the parameters were set as default. The top-scored model was chosen and then submitted to verify using the SAVES v5.0 server (https://servicesn.mbi.ucla.edu/SAVES/).The protein sequence of human SPT (UniProtKB: O15269) was retrieved from the NCBI database (2.2.https://pubchem.ncbi.nlm.nih.gov/). The Specs and Chemdiv databases were prepared by the Prepare Ligands protocol in DS3.5. And then, the Find Similar Molecules by Fingerprints module was employed with the Tanimoto coefficientThe 3D structure of myriocin was retrieved from the Pubchem database  was used as the initial receptor of subsequent molecular docking virtual screening. The protein was prepared using the Prepare Protein protocol in DS3.5, to remove all crystallographic water, add hydrogen atoms, repair broken chains, and add CHARMm force field. Afterward, a sphere binding site was generated with the co-crystallized myriocin as centroid using the define site tool in DS3.5.Molecular docking: The CDOCKER protocol in DS3.5 was used to perform the VS. First, the prepared 4BMK was submitted to screen the similarity molecules, the top hits were set to 5 and the other parameters were set as default. Afterward, the top-scored pose of each similarity molecule was retained, and all selected docking poses were submitted to VS with the modelling-human SPT protein.2.4.The synthesis of compoundWXP-001: l-Serine  was dissolved in 80 ml of toluene, and dodecanol , p-toluenesulfonic acid monohydrate  in turn were added to above solution at room temperature. Then the oil–water separator was connected and heated to 110 °C under reflux overnight. The reaction was monitored by TLC. The reaction solution was cooled to room temperature and diluted with ethyl acetate. Then the organic phase was washed with saturated sodium bicarbonate and brine successively and dried with anhydrous sodium sulphate. After filtered, the filtrate was concentrated in vacuo through a silica gel column (PE:EA = 1:1–1:3∼DCM:MeOH = 20:1) to obtain 2.5 g of brown oil l-serine lauryl ester. The resultant brown oil (800 mg) was dissolved in 5 ml mixture solution containing hydrogen chloride and methanol, stirring at room temperature for 15 min, and concentrated under vacuum to obtain the product l-serine dodecyl ester hydrochloride , light brown particles. LC-MS(ESI): 274.4 (M + H)+, RT = 4.75 min; 1H NMR  δ 8.43 , 5.59 , 4.19–4.11 , 3.86–3.77 , 1.64–1.57 , 1.32–1.25 , 0.88–0.84 .The synthesis of compoundWXP-002: l-Leucine  was dissolved in toluene (20 ml), then added octadecan-1-ol  and p-toluenesulfonic acid  to above solution. Use the method of synthesis of WXP-001. Purified through silica gel chromatography (PE:EA = 2:1) to give yellow oily leucine octadecy ester . LC-MS(ESI): 384.5 (M + H)+, RT = 6.68 min; 1H NMR  δ 8.48 , 4.20–4.09 , 3.97–3.93 , 1.76–1.72 , 1.64–1.58 , 1.29–1.23 , 0.91–0.89 , 0.87–0.83 .The synthesis of compoundWXP-003: l-Leucine  was dissolved in toluene (20 ml), then added dodecanol  and p-toluenesulfonic acid  to above solution. Use the method of synthesis of WXP-001. Purified through silica gel chromatography (PE:EA = 2:1) to give yellow oily leucine dodecyl ester . LC-MS(ESI): 300.3 (M + H)+, RT = 2.94 min; 1H NMR  δ 4.12 , 3.46 , 1.83 , 1.77–1.75 , 1.74–1.72 , 1.45–1.20 , 0.95–0.83 .2.5.Aspergillus fumigates (CMCCC(F) A.1a), Cryptococcus neoformans (CMCCC(F) D.2a), Sporothrix (CMCCC(F) D.1a), Rhizopus oryzae (CMCCC(F) B.81a), Candida parapsilosis (ATCC22019) and Candida krusei (ATCC6258). Myriocin was prepared to 2 mg/mL solution in methanol and caspofungin was prepared to 5 mg/mL solution in water. Compounds WXP-001∼003 were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions of 10 mg/mL concentration.MIC determinations were finished via broth microdilution assay with myriocin and caspofungin used as positive inhibitor controls. All experimental fungus were provided by the Pathogenic Microorganism (toxin) Preservation Center of Chinese Academy of Medical Sciences, including four species of fungi and two drug-sensitive reference strains as follows: 3 cfµ/mL and filamentous fungus density of 0.4–5 × 104 cfµ/mL. All the plates were incubated at 37 °C. The OD value of each well was recorded, the blank subtracted out, and the growth in each well calculated as percentage of the control. The lowest concentration of compound that resulted in <10% growth was recorded as the MIC.The compounds along with positive controls were serially diluted to 10 concentrations solution across the 96-well plates, ranging from 100 µg/mL to 0.195 µg/mL with final volumes of 100 µL per well. Yeasts were cultured in Sabouraud Dextrose Agar (SDA) at 30 °C for 48 h. Filamentous fungus were cultured in Potato Dextrose Agar (PDA) at 26 °C for 7–10 d. Each sample was then diluted to suspension with sterile saline solution and was calculated the density. The resultant suspensions were diluted and added to above compound-containing 96-well plates (100 µL per well) to give the yeasts density of 0.5–2.5 × 102.6.Cells in the logarithmic phase were removed to a centrifuge tube and sonicated for 30 min in an ice bath. Fungi were also treated under same conditions. Collected 1.0 ml of supernatant by centrifugation, then added 2.0 mL of extraction mixture (isopropanol: ultrapure water: ethyl acetate = 30:10:60). The mixture was extracted for 2 h at 180 rpm and separated by standing or centrifugation, and the supernatant was taken as the test sample. Diluted the standard and determined the standard curve. Set blank well and sample well, added 40 µL of sample diluent to the 96-well plates, and added 10 µL of sample to be tested . After sealed with the sealing film, the plates were incubated at 37 °C for 30 min. Diluted the 30-fold concentrated solution with distilled water 30-fold before use. Then each well was washed with washing liquid for 5 times, each time for 30 s. Except for blank wells, sample wells were added 50 µL of enzyme-labelled reagent. Repeated the incubation operation and washed. Each well was added a colour developer and mixed gently, then incubated at 37 °C for 10 min in the dark. After incubation, added 50 µL stop solution to stop the reaction. OD value was measured at 450 nm.2.7.WXP-003 complex, with the best bioactivity, generated by molecular docking was used as initial structures to carry out MD simulation by Desmond software package+/Cl− counter ions. Further, the solvated system was minimised under the OPLS_2005 force field. Afterward, the complex was submitted to a 60-ns MD simulation following the default NPT and NVT ensemble protocols in Desmond program. During the production run, the temperature and the pressure were set to 300.0 K and 1.01325 bar, respectively. The energy and atomic coordinate trajectory recording interval were set to 20 ps. After MD simulation, The RMSD and protein-ligand contacts were calculated with the Simulation Interaction Diagram protocol in Desmond packageThe SPT/3.3.1.in silico screening employed in this study involves two stages of establishment of homology modelling and docking-based virtual screening.The procedure of wittichii), 2JG2 , 3A2B  and 4BMK . As is well known, the sequence alignment plays a key role in homology modelling method. From the BLASTP results, the human SPT exhibited a good alignment with the selected template proteins. The sequence identities were 29.41% for 2X8U, 28.44% for 2JG2, 32.33% for 3A2B, and 28.44% for 4BMK, and the alignments of structures and sequences showed in Initially, the input human SPT sequences were searched to find homologous protein structures using BLAST, and this resulted four proteins as matching proteins in the PDB, structure ID 2X8U  and three clinical drug-resistance fungal strains  were used as tested fungi. These compounds were initially screened to inhibit fungi growing at the concentration of 100 µM, 50 µM and 10 µM. Only 5 compounds displayed promising anti-fungal growing activity against diversity fungi at tested concentration (Supplementary Table S1). WXP-004∼005 only showed low activity against some kinds of fungi. Especially, compounds WXP-001∼003 showed significant anti-fungal activity against the tested fungi, the structures as shown in WXP-001∼002 inhibited all the kinds of tested fungi growing at the concentration of 50 µM, while WXP-003 revealed the brilliant inhibitory activity against all the tested fungal strains at 10 µM (Supplementary Table S1). Therefore, WXP-001∼003 were selected to further evaluated on more kinds of fungi to detect the MIC value in detail.The 82 compounds screened WXP-001 was synthesised through ester condensation reaction with l-serine and dodecanol as the start materials. Compounds WXP-002∼003 were employed the same method of WXP-001. Notably, all the products were needed to be stirred in mixture solution of hydrogen chloride and methanol to produce hydrochloride product. All the steps obtained moderate yields.Based on above preliminary screening on anti-fungal activity, we first synthesised the three potential compounds to make sure enough amounts of compounds for the further evaluation. As shown in WXP-001∼003 exhibited fungicidal activity against six fungal strains evaluated in the test of the minimum inhibitory concentration (MIC) with myriocin and caspofungin used as control, these fungi including A. fumigatus, R. oryzae, cryptococcus, sporothrix, C. krusei and C. parapsilosis (WXP-001 only displayed more outstanding inhibitory activity (MIC = 3.125 μg/mL) against C. krusei than caspofungin (MIC = 25 μg/mL), low inhibitory activity against A. fumigatus, R. oryzae and sporothrix. Compound WXP-002 exhibited moderate activity against all the tested fungi strains with MIC value ranging between 10 and 100 μg/mL, but obviously lower than positive control. Significantly, WXP-003 showed the highest inhibitory activity against all the tested fungal strains during these three compounds. Anti-fungal activity of WXP-003 against C. krusei and R oryzae was superior with MIC value at 0.78 μg/mL and 12.5 μg/mL, while caspofungin only showed MIC value at 25 μg/mL and 100 μg/mL. Similarly, compound WXP-003 exhibited better activity against A. fumigatus and cryptococcus  than SPT inhibitor myriocin .Here, compounds psilosis . CompounWXP-003 was selected to test the effect on sphingolipids content in fungi and mouse fibroblast cell line L929, myriocin as positive control and vehicle as negative control . WXP-003 could significantly reduce sphingolipids content in fungi and no effect on mammal cells.Thus, compound  control . The resWXP-003 presented the potent anti-fungal activity in this study and then we further explored the molecular dynamics simulation of WXP-003 with fungi SPT to help us understand the binding mode of WXP-003 and fungi SPT.In short, 3.3.WXP-003 complex, MD simulation was conducted. First, the stability of the system was evaluated and monitored through the RMSD (root-mean-square deviation) of the backbone atoms. As shown in Supplementary Figure S1, during the last 30 ns, the RMSD values were considerably stable, suggesting that the MD simulation reached equilibrium within the last 30 ns.To further investigate the dynamic interaction patterns of SPT/WXP-003, the per-residue contributions in ligand binding were calculated. WXP-003 could well adapt to the binding pocket of SPT, which was similar to the myriocin/SPT complexWXP-003 covalent bond to the PLP , and the SPT/WXP-003 complex also formed the favoured interactions with the key residues within the active site, such as Gly134, His159, Lys265 11,27.3.4.WXP-003 was below 500 and the log P values lie in the accepted limits. WXP-003 had shown 3 hydrogen bond acceptors and 2 hydrogen bond donors.The correlation between physio-chemical properties of the chemical moieties and the drug pharmacokinetics in the human body was calculated by the Lipinski rule of five. Despite some exception in drug discovery, it is a widely accepted method for preliminary screening of drugs. The results deduced from the molinspiration server are summarised in 4.WXP-003 was screened through in silico and in MIC assays and was synthesised. WXP-003 showed strong inhibitory activity against the tested fungal strains. WXP-003 could significantly reduce sphingolipids content in fungi and no effect on mouse fibroblast cell L929. Meanwhile, molecular dynamics simulation was conducted to investigate the dynamic interaction patterns of SPT/WXP-003 complex, and then the SPT/WXP-003 complex formed the favoured interactions with the key residues within the active site. The prediction of physio-chemical properties provided further evidence that WXP-003 may be a promising anti-fungal agent. This work offers a new potential approach based on discovering selective fungi SPT inhibitor.In summary, we have constructed homology models of human serine palmitoyltransferase (SPT) for virtual screening. A logical docking-based virtual screening approach was employed to identify novel fungi SPT inhibitors while except human SPT inhibitors. In our study, Click here for additional data file.


The mission of applied gerontology is to bridge science and practice to benefit the health and well-being of older persons, their families, their communities, and other contexts. This presentation will provide insights from the Journal of Applied Gerontology and its attempts to publish and disseminate scholarship that has international application. Following an overview of the growing internationalization of peer-reviewed submissions to the Journal of Applied Gerontology on a variety of topics and from a range of perspectives, the presentation will highlight key achievements as well as ongoing concerns and opportunities to better achieve the goals of applying gerontological scholarship to aging contexts worldwide. Concluding comments will examine how outlets for dissemination and authors themselves can better position their work to enhance their influence on aging in an international context.