Depression is characterized by the disruption of both neural circuitry and neurogenesis. Defects in hippocampal activity and volume, indicative of reduced neurogenesis, are associated with depression-related behaviors in both humans and animals. Neurogenesis in adulthood is considered an activity-dependent process; therefore, hippocampal neurogenesis defects in depression can be a result of defective neural circuitry activity. However, the mechanistic understanding of how defective neural circuitry can induce neurogenesis defects in depression remains unclear. This review highlights the current findings supporting the neural circuitry-regulated neurogenesis, especially focusing on hippocampal neurogenesis regulated by the entorhinal cortex, with regard to memory, pattern separation, and mood. Taken together, these findings may pave the way for future progress in neural circuitry–neurogenesis coupling studies of depression. Depression is the leading cause, only second to heart diseases, of disability worldwide . The gloHippocampal neurogenesis defects are a hallmark of depression. Mounting evidence points to hippocampal deficits in activity and volume and reductions in activity-dependent gene expression, which collectively reflect neurogenesis defects in the dentate gyrus of the hippocampus ,10,11. HOur formulation of the neural circuitry–neurogenesis coupling model of depression is based on both long-lasting neurophysiological knowledge of adult hippocampal neurogenesis and pioneering works not just recapitulating neurogenesis but also demonstrating the impact of neural circuitry modification on neurogenesis and depression-related behaviors. Here, we review the neurophysiological findings supporting that the entorhinal cortex and hippocampus collectively contribute to adult neurogenesis. The entorhinal cortex and hippocampus are synaptically involved in adult neurogenesis ,21,22,23Episodic memory damage is a major cognitive symptom of depression ,38,39,40Entorhinal–hippocampal circuitry is known as a memory hub of human and primate brains, mainly in processing episodic memories of objective, spatial, and temporal information, such as what, where, and when ,53,54,55In addition to this finding in humans, mouse studies also demonstrated that deep brain stimulation of the entorhinal cortex leads to improvements in spatial learning and memory that are accompanied by enhanced neurogenesis in the dentate gyrus b. SpecifIn addition to the deep brain stimulation approach, preclinical research has employed an optogenetic approach to gain further insight into the detailed physiology of memory implicated in entorhinal–hippocampal circuitry. Robinson and colleagues questioned whether entorhinal–hippocampal circuitry regulates temporal memory that is encoded in the principal cells, also known as time cells, of the hippocampal CA1 region . They inFurther research is required to address these questions: which specific cell types, or neurotransmitters, of the entorhinal cortex and hippocampus are implicated in spatial and temporal memory encoding defects in stress animal models, respectively; whether temporal memory encoding in the hippocampus still relies on neurogenesis; how the entorhinal cortex drives specific electrophysiological changes, such as hippocampal theta rhythm oscillations, in association with long-term potentiation of spatiotemporal memory during neurogenesis in stressed animals and how the rhythm changes are associated with memory deficits in the depression models; and ultimately, whether therapeutic targeting of the entorhinal–hippocampal circuitry using brain stimulation or pharmacological approaches recovers the spatiotemporal memory deficits with substantial validity and reliability, specifically in humans with depression.Pattern separation is the ability to distinguish between similar contextual representations and is dependent on hippocampal dentate gyrus neurogenesis ,68,69,70Functional imaging studies raise the possibility that the entorhinal cortex may be implicated in the upper hippocampal circuitry to operate pattern separation. Earlier studies used elderly human subjects to examine the association between the entorhinal cortex-implicated circuit dysfunction and cognitive declines, because volume reductions in medial temporal lobes including entorhinal cortex have been found to relate to cognitive deficits in both depression  and aginAmong the earliest approaches in the study of the impact of neurogenesis defects on pattern separation, hippocampal X-ray irradiation or the disruption of synaptic plasticity in dentate granule cells was mostly adopted to recapitulate neural stem cell niche dysfunction to scrutinize impairment in the discrimination of a safe, similar context from the foot-shock context ,81,82,83Trip8b, a knockdown of which is known to increase the excitability of hippocampal neurons, enabling dentate gyrus neurogenesis. In the knockdown mice, stimulation of entorhinal glutamatergic afferents led to activity-dependent hippocampal neurogenesis, including both the generation and dendritic maturation of adult-born dentate gyrus neurons through enhanced intrinsic excitability of stellate cells in the entorhinal cortex. Then, a behavioral task using contextual fear conditioning was adopted to examine the knockdown mouse’s ability (pattern separation) to discriminate the foot shock-associated context from a safe, similar context. The knockdown mice (Trip8b-shRNA) exhibited approximately 50% more freezing in the foot shock-paired context compared with the control mice (SCR-shRNA), thus revealing enhanced pattern separation from glutamatergic entorhinal stimulation. Furthermore, dentate gyrus-directed image-guided X-ray irradiation, reflecting neurogenesis ablation, significantly blunted the effect induced by entorhinal-specific Trip8b knockdown. Together, this pioneering work using a chemogenetic approach supports the idea that entorhinal–hippocampal circuitry regulates neurogenesis, thereby enabling pattern separation. Nonetheless, our understanding of the circuitry-regulated neurogenesis mechanism underlying pattern separation remains rudimentary; thus, this field of preclinical research requires more efforts to exploit brain stimulation and optogenetic and chemogenetic approaches to delineate the mechanistic comprehension of entorhinal–hippocampal circuitry giving rise to pattern separation.Yun and colleagues adopted a chemogenetic approach to develop a transgenic mouse model with entorhinal cortex-specific knockdown of a psychosocial stress-induced protein using adeno-associated virus-mediated gene transfer c 19]. A. A19]. AHippocampal neurogenesis is closely linked to the pathophysiology of depression as well as the response to antidepressant treatments ,85. HippTo date, there are no available studies that exploit deep or superficial brain stimulations targeting the entorhinal cortex to conceptually link hippocampal neurogenesis and anti-depressive behaviors. Most studies have focused on other brain regions, including the prefrontal cortex ,106,107,Trip8b, a specific stress-induced protein that affects hippocampal neuron activity. Indeed, Trip8b germline knockout mice demonstrated increased hippocampal neuron firing frequency and more neurogenesis and new neuron maturation than controls, particularly in the temporal dentate gyrus, which is a hippocampal subregion associated with emotion processing and response to stress valences . Th. Th19]. valences ,134. Theosterone ,136. Undpression  exclusivpression . These fPioneering preclinical studies linking entorhinal–hippocampal circuitry, adult neurogenesis, and emotional and cognitive symptoms of depression proposed a first step for the neural circuitry–neurogenesis coupling model in depression. Nonetheless, more efforts should be made to elucidate the mechanism of the depression model in two parallel directions. Firstly, future research is required to delineate the entorhinal–hippocampal circuitry in detail, considering the complex neurochemical physiology between the entorhinal cortex and hippocampal dentate gyrus. During adult neurogenesis, the entorhinal cortex provides major glutamatergic afferents to the hippocampal dentate gyrus, through which the progeny cells of the hippocampus mature to become granule cells that can be integrated into hippocampal circuits ,140,141.Secondly, future research is also required to elucidate the complex upper hippocampal circuitries that encompass diverse brain regions implicated in depression pathophysiology while maintaining the perspective of hippocampal neurogenesis. Novel evidence suggests that brain regions beside the entorhinal cortex are also related to dentate gyrus neurogenesis, suggesting that complex neural circuitries are implicated in hippocampal-dependent functions as well . For exaAs shown in


Adenomatous Polyposis Coli (APC). APC mutation is thought to allow a “just-right” amount of Wnt pathway activation by fine-tuning β-catenin levels. While at a much lower frequency, mutations that result in a β-catenin that is compromised for degradation occur in a subset of human CRCs. Here, we investigate whether one such “stabilized” β-catenin responds to regulatory stimuli, thus allowing β-catenin levels conducive for tumor formation. We utilize cells harboring a single mutant allele encoding Ser45-deleted β-catenin (β-catΔS45) to test the effects of Wnt3a treatment or APC-depletion on β-catΔS45 regulation and activity. We find that APC and β-catΔS45 retain interaction with Wnt receptors. Unexpectedly, β-catΔS45 accumulates and activates TOPflash reporter upon Wnt treatment or APC-depletion, but only accumulates in the nucleus upon APC loss. Finally, we find that β-catenin phosphorylation at GSK-3β sites and proteasomal degradation continue to occur in the absence of Ser45. Our results expand the current understanding of Wnt/β-catenin signaling and provide an example of a β-catenin mutation that maintains some ability to respond to Wnt, a possible key to establishing β-catenin activity that is “just-right” for tumorigenesis. The Wnt/β-catenin signaling pathway is deregulated in nearly all colorectal cancers (CRCs), predominantly through mutation of the tumor suppressor  Discovered nearly 40 years ago, the Wnt signaling pathway has proven essential for many cellular functions, including proliferation, polarity, developmental cell-fate determination, and tissue homeostasis . ConsequThe key downstream effector molecule in the Wnt pathway is the transcription cofactor β-catenin. In the absence of a Wnt signal, a cytoplasmic β-catenin destruction complex efficiently catalyzes the proteasome-mediated degradation of β-catenin. The core components of the complex include two scaffolding proteins, Adenomatous Polyposis Coli (APC) and Axin, as well as two kinases, GSK-3β and CK1-α . In the β-TrCP which ubiquitinates β-catenin and marks it for degradation by the proteasome [In canonical Wnt signaling, β-catenin destruction is initiated through a dual-kinase mechanism. First, CK1-α phosphorylates Ser45 of β-catenin A,B 8,11,118,11. oteasome ,16,17,18Adenomatous Polyposis Coli (APC) occur early in the development of over 80% of CRCs. The vast majority of APC mutations lead to the expression of truncated APC protein that retains some ability to interact with and regulate β-catenin. The “just right” model rationalizes the limited range of APC truncations observed in CRCs as facilitating a precise level of β-catenin for optimal cellular proliferation—not too much or too little [Mutations in the tumor suppressor o little . In addio little ,22,23,24o little ,26. Addio little ,27,28. Ao little ,28.APC mutations commonly have mutations in genes encoding other components of the Wnt pathway. The key downstream effector molecule in Wnt signaling, the β-catenin gene, CTNNB1, is mutated in ~12% of CRCs lacking an APC mutation  cells, Rebouissou and colleagues reported that S45 mutations are only weakly activating and they concluded that S45 mutation alone is not sufficient to drive liver tumorigenesis [Axin2, GLUL, and LGR5, but not to the levels seen in patients with mutations in D32-S37  and found that β-catenin mutations occurred in 160/2324 of patients  dataset for expression of Wnt target genes in colon cancers with various categories of ild-type A. TherefCTNNB1 status . To ensure that mutations in these other β-catenin regulators were not influencing Wnt target gene expression in our “wild-type” samples presented in AXIN2 and LGR5 were either unchanged or decreased in patients with CTNNB1 mutations when compared to patients that were wild-type for all of these β-catenin regulator genes. Though these results were consistent with our in vitro data, they contradict results from a similar analysis of liver cancers [CTNNB1 mutations are not the same in the liver and colon.Using a similar approach, sample groups were established based on 1 status B. There  cancers  and  Wnt signaling leads to a secondary effect that further activates β-catenin in the nucleus, essentially amplifying the signal. An example of this would be that get gene ,50. (2) CTNNB1 mutation are not the same in cancers of different tissues. Our work is in agreement with previously published results by our lab and others, demonstrating that the interaction of APC with nuclear β-catenin leads to repression of Wnt target genes ,21,23,24APC-mutant cells are unresponsive to additional Wnt signaling, whereas cells expressing mutant β-catenin are responsive to extracellular Wnt ligand. It is likely that complete stabilization of β-catenin in colon epithelia would result in too much β-catenin protein and activity, thus impairing cell viability and potentially inducing apoptosis as previously demonstrated in mouse gut [Colorectal cancer is generally assumed to be promoted by Wnt signal activation. Here, we provide insight into the mechanism by which β-catenin is regulated in the Wnt-on or Wnt-off states and also demonstrate that mutant β-catenin can be regulated by the Wnt pathway components. We find that ouse gut  and humaouse gut . Our finKO cells were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) and were maintained at 37 °C and 5% CO2. Wnt treatment was performed by adding recombinant Wnt-3a  at the indicated concentration/time prior to cell lysis or immunofluorescence analysis. For siRNA-mediated inhibition, HCT116βm cells were transfected using Lipofectamine 3000  according to the manufacturer’s instructions with 37.5 nM of each siRNA targeting human APC  or nontargeting siControl siRNA (Dharmacon). Cell media was changed one day following siRNA transfection, and cells were grown 48 h prior to Wnt treatment. MG-132 treatment was performed by the addition of 10 mM MG-132 to a final concentration of 10 μM in cell media for 4 h.HCT116βm, RKO, DLD1, and RKO-APCKO cells were derived from the parental RKO cell line (ATCC) and kindly provided by Ethan Lee [HCT116βm were derived from the parental HCT116 cell line  and kindly provided by Bert Vogelstein . We recethan Lee . We passCTNNB1 gene was assessed using the cBioPortal [CTNNB1 and APC genes [CTNNB1 mutations or “driver” mutations in APC or other β-catenin regulatory factors. Values were provided as RSEM (RNA-Seq by Expectation Maximization).Mutation frequency of the ioPortal ,36. ColoPC genes ,38,39,54HCT116βm and RKO cells were harvested and DNA extraction was performed using the Qiagen  DNeasy Blood and Tissue Kit according to the manufacturer’s instructions. PCR amplification was performed using the following primers: 5′-cctcctaatggcttggtgaa-3′; 5′-caggacttgggaggtatcca-3′. Following amplification, PCR products were gel-purified and sequenced by Genewiz . Trace files and sequence alignment were analyzed using SnapGene, version 4 (Insightful Science). N-morpholino)ethanesulfonic acid (MES) buffer. Wnt immobilization was performed by diluting 0.5 μg of purified Wnt3a protein in cold MES buffer and incubated at room temperature (RT) for 1 h. To quench nonreactive carboxylic acid groups, beads were incubated with 50 mM Tris pH 7.4 at RT for 15 min. Beads were washed twice in phosphate-buffered saline (PBS) pH 7.4 before final resuspension in 400 μL PBS/0.5% BSA and stored at 4 °C. Unloaded-beads were prepared in parallel by incubating 1 h in MES without Wnt. Wnt3a activity following bead immobilization was verified using a TOPflash luciferase reporter assay [Wnt3a was immobilized onto Dynabeads as described previously . Brieflyer assay . Cells were washed 1x in PBS prior to harvesting in preheated, high-salt sample lysis buffer , 30% 10X PBS, 2.5% β-mercaptoethanol). Scraped cells were transferred to Eppendorf tubes, heated at 95 °C for 1 min, pulled through an insulin syringe three times, and heated again. Samples were separated on 7.5% SDS-PAGE  using Tris-Glycine running buffer and transferred to a nitrocellulose membrane (GE) with a 0.45 μm pore size. Antibodies were diluted in Odyssey Blocking Buffer TBS (LI-COR) as follows: anti-APC-M2 Chicken pAb (1:2000), anti-β-catenin mouse mAb (1:1000), anti-phospho-Ser33/Ser37/Thr41-β-catenin rabbit pAb , anti-α-tubulin DM1A mouse mAb , anti-β-actin mouse mAb , and IRDye 680LT and 800CW anti-rabbit, anti-mouse, or anti-chicken secondary antibodies . Immunoblots were imaged on an LI-COR Odyssey CLx imaging system. Cells were grown in 6-well tissue culture plates and treated with 40 μL Unloaded-beads or Wnt-beads for 4 h. Following bead treatment, cells were briefly washed in 1x PBS prior to lysis in 200 μL lysis buffer  ,26. FollE. coli cells (NEB) for expression. Cells were grown in standard LB broth containing 50 μg/mL kanamycin at 37 °C with shaking (225 rpm) and induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression when an OD of 0.4–0.6 was reached. Cells were allowed to express induced protein products for 3–4 h at 37 °C with shaking before harvested by centrifugation at 4000 rpm, 15 min, 4 °C. Cellular pellets were resuspended in a buffer containing 50 mM Tris pH 8.0, 50 mM NaCl, 50 mM imidazole, 10% glycerol, and HALT protease cocktail (Thermo). Cells were lysed by a French pressure cell , and the insoluble cellular debris was removed by centrifugation at 16,000× g for 45 min, 4 °C. Supernatant was applied to a chelating sepharose fast-flow column  charged with nickel chloride and pre-equilibrated in resuspension buffer. Protein retained on the column was washed with a 3-column volume (C.V.) salt gradient . Protein was eluted with an imidazole buffer gradient . Fractions containing recombinant protein were pooled and applied to a Superdex 200 size-exclusion column (Amersham Biosciences) pre-equilibrated with 50 mM Tris, pH 8.0, 150 mM NaCl, and 10% glycerol, 1 mM DTT, and 1 mM octyl β-D-gluctopyranoside. Fractions containing recombinant protein were pooled and concentrated with Amicon Ultra centrifugal filters (Millipore). The central region of APC  was cloned and ligated into the pET28b  expression vector, which contains an N-terminal 6X-His tag. Sequence-verified plasmids were transformed into BL21-CodonPlus-(DE3)-RIPL Recombinant APC antigen was shipped to Gallus Immunotech Inc.  for injection into hens, and extraction of IgY-containing yolk extract. The anti-APC IgY was purified from the returned yolk extracts by affinity chromatography. Briefly, NHS-Sepharose  was conjugated with APC-M2 antigen, purified as described above. 1M Tris (pH 8.0) and Tween-20 were added to the yolk extracts, to final concentrations of 10 mM and 0.2%, respectively. Buffered yolk extract was incubated with prepared APC-M2-conjugated NHS-Sepharose overnight at 4 °C. Columns were washed with multiple column volumes of PBS and PBS-T buffers. Anti-APC-M2 IgY was eluted from the column in 0.2 M glycine, pH 2.0. Fractions containing eluted anti-APC-M2 IgY were dialyzed into a buffer containing PBS at pH 7.4, 5% glycerol, and 0.01% sodium azide. Cells were seeded into 12-well plates 24 h prior to transfection. On the day of transfection, cell culture media was changed one hour prior to siRNA and plasmid transfection. Both reporter plasmids and siRNAs were transfected concurrently using Lipofectamine 3000 according to the manufacturer’s instructions. Human APC-siRNA or control-siRNA was transfected as described above. For luciferase reporters, cells were cotransfected with TOPflash (450 ng) and Renilla (50 ng) expression plasmids. As a control, identical wells were transfected with FOPflash (450 ng) and Renilla (50 ng) expression plasmids to validate that a scrambled TCF-binding sequence does not result in increased reporter activity upon treatment. Following two days of APC-depletion, cells were treated with Wnt3a for 24 h and lysed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase signal was normalized to the Renilla luciferase signal, and data were normalized to control (set to 1). 2, 1 mM EGTA) for 20 min at room temperature (RT) and washed two times in PBS prior to permeabilization in TBS + 0.2% Triton X-100 for 5 min. Cells were washed in TBS two times prior to incubation for 1 hour at RT in blocking buffer containing TBS + 0.2% Triton X-100, 1% BSA, and 3% Normal Goat Serum. Primary and secondary antibodies were incubated for 1 hour at RT. Cells were washed three times in TBS following primary and secondary antibody incubations. Mounting of coverslips was performed using Prolong Diamond Antifade Mountant with DAPI (Invitrogen). The anti-β-catenin mouse mAb  was diluted 1:250 in blocking buffer. Stained cells were examined using an Axioplan microscope  with a ×100 objective. Images of stained cells were captured using an Orca R2 digital camera . Cells were briefly rinsed in PBS prior to fixation. Cells were fixed in 4% PFA in Brinkley’s Buffer 1980 . Graphs were generated using GraphPad Prism, version 8.4.3 , and all statistical analyses were performed using a two-tailed, unpaired t-test in which a value of APC mutations occur in a “just-right” fashion to allow optimal levels of β-catenin signaling for tumorigenesis. The current work demonstrates that stabilizing β-catenin mutations allow cells to respond to Wnt and are prone to regulation by APC. Wnt treatment and APC-depletion both induce increased β-catΔS45 protein and activity by the TOPflash reporter assay. Interestingly, we find that Wnt treatment does not increase the nuclear accumulation of β-catΔS45 whereas APC-depletion results in predominantly nuclear β-catΔS45 localization. This data supports a role for APC in cytoplasmic retention of β-catenin. Further, we verify that β-catΔS45 can be phosphorylated at the GSK-3β phosphorylation sites, despite the lack of a priming Ser45. Treatment with proteasome inhibitors results in β-catΔS45 accumulation, indicating that this “stabilized” protein is still able to be targeted to the proteasome. Our results indicate that a Wnt signal leads to secondary effects that further activate nuclear β-catenin and that APC may inhibit this activity by nuclear export and/or sequestration of β-catenin in the cytoplasm. Using data available on cBioPortal, we find that mutations to β-catenin occur in ~6% of human CRC and in 9% of CRCs lacking an APC mutation. Combined with our previously published data demonstrating that the β-catenin destruction complex relocalizes toward a Wnt cue in cells expressing mutant β-catenin, we hypothesized that “stabilizing” β-catenin mutations do not completely evade Wnt regulation. It has been previously proposed that


While seminal theories suggest that nonsymbolic visual numerosity is mainly extracted from segmented items, more recent views advocate that numerosity cannot be processed independently of nonnumeric continuous features confounded with the numerical set . To disentangle these accounts, here we employed two different visual illusions presented in isolation or in a merged condition . In particular, in a number comparison task, we concurrently manipulated both the perceived object segmentation by connecting items with Kanizsa-like illusory lines, and the perceived convex-hull/density of the set by embedding the stimuli in a Ponzo illusion context, keeping constant other low-level features. In Experiment 1, the two illusions were manipulated in a compatible direction , whereas in Experiment 2 they were manipulated in an incompatible direction . Results from psychometric functions showed that, in the merged condition, the biases of each illusion summated  in Experiment 1, while they averaged and competed against each other in Experiment 2. These findings suggest that discrete nonsymbolic numerosity can be extracted independently from continuous magnitudes. They also point to the need of more comprehensive theoretical views accounting for the operations by which both discrete elements and continuous variables are computed and integrated by the visual system.The online version contains supplementary material available at 10.3758/s13423-021-01979-w. Main computational, psychophysical, and neuroimaging studies  from continuous visual features confounded with numerosity  therefore represents the main theoretical and experimental challenge to probe which visual mechanisms and sensory features are exploited by the ANS to reach an approximate numerical representation .By contrast, other studies manipulating the perceived size of continuous features by means of size illusions bring evidence in favor of the indirect account  one type of information  prevails over the other or (ii) both types of information independently contribute to numerosity perception. To this aim, in the present study, we concurrently applied two different visual illusions over the same stimuli to shed light on the processing of visual discrete numerosity information and continuous physical features. A similar approach, combining visual illusions in the same stimulus, has been already employed to investigate the extent to which different simultaneous visual distortions may interact affecting the final percept, a condition not uncommon in real-world perception and in visual arts such as drawing , one condition with only the Kanizsa illusion, one condition with only the Ponzo illusion, and one combined (or merged) condition, with illusions triggering a bias in the same direction . If numerosity is processed independently from continuous magnitudes, we should find the larger underestimation in the combined condition compared with the single illusion conditions. On the contrary, according to the indirect account, the bias in the combined condition should not differ from the bias in the condition with only the Ponzo illusion, as the perceived convex hull/density should play the leading role in driving numerosity estimation.www.pavlovia.org), a repository and launch platform allowing online experiments. A total sample of 67 participants  took part in the study. All participants had normal or correct-to-normal vision and were naïve about the purpose of the experiment. The study was approved by the Local Ethical Committee (protocol N° RM-2020-230).Due to COVID-19 restrictions in Italy, the participants were recruited through Pavlovia , consisting of 12 black “Pac-Man”-like items  spatially scattered and randomly rotated at an angle varying across 360° to avoid collinearities and pop-out of illusory contours (ICs). The test patterns contained a variable numerosity, that is, from nine to 15 “Pac-Man”-like items. Half of the test patterns (N = 84) were composed by “Pac-Man”-like items that were not eliciting any ICs, while in the other half of test patterns (N = 84) “Pac-Man”-like items were purposely aligned to prompt ICs .The whole experimental set was composed of 168 test patterns  and of 168 reference patterns . The reference patterns always contained the same numerosity , which were coupled to the 42 reference patterns. In each test pattern of the no-illusions condition set, all the inducers were not aligned (did not trigger ICs). Each of the 42 stimuli pairs of the no-illusions condition  were embedded inside two parallel black lines  forming a rectangle whose base was 580 pixels and the height 250 pixels placed at the screen center. These two parallel lines did not elicit any illusion (and were used as a control for the Ponzo illusion).In the Kanizsa illusion condition, to keep constant spatial profiles of test sets from the baseline , each different test pattern for each numerosity of the no-illusions condition was cloned. Thus, we kept constant the spatial position of all the single items in a given test pattern from the no-illusions set. Critically, in this case a subset of “Pac-Man” items was appropriately rotated and aligned to prompt four ICs for the Kanizsa condition. The distance between the “Pac-Man” items that could prompt the required number of ICs for the connectedness (or Kanizsa) condition was randomly chosen among four possible values . In this way, the 42 different reference patterns were associated with the same spatial pattern of test stimuli across the no-illusions and the Kanizsa illusion condition. In both conditions, test and reference stimuli were embedded inside two parallel black lines, so that no Ponzo illusion was prompted.Then, these two conditions were cloned and drawn embedded in the Ponzo illusion context, thus generating stimuli pairs for the Ponzo illusion condition and the combined Ponzo–Kanizsa illusion condition. The Ponzo illusion was elicited by two tilted black lines  forming the legs of an isosceles trapezoid whose virtual longer base was 300 pixels length and whose shorter base was 250 pixels length (distance between the bases of 580 pixels). Note that in the experiment, the relative positions of the reference and the test stimuli were randomized between the left and right side. Yet the test set was always anchored in the larger part of the Ponzo illusion context, which was randomized in accordance with the position of the test stimulus, so that when the test stimulus appeared to the right, the Ponzo illusion context was drawn with the larger side on the right side.All the patterns in the four experimental conditions were drawn on a grey background  and reference and test stimuli were projected within two virtual squared panels (240 × 240 pixels) centered at ±156 pixels from the screen center. Furthermore, we constrained the single “Pac-Man” items in test and in reference stimuli to be distant at least 20 pixels from the four virtual square edges and to not overlap with each other (minimum center-to-center distance = 22 pixels).The stimuli were presented by means of an online PsychoPy routine Peirce, , and allThe participants performed a two-alternative forced-choice task, in which they were asked to choose the set containing more dots between two rapidly presented visual patterns by pressing the corresponding keys on the keyboard. The experimental phase was preceded by a brief training composed of 24 trials  to allow the subject to familiarize with the task. In the training phase, we presented only the reference patterns versus the test pattern with nine items. Each experimental trial started with a middle-grey background  lasting 1,000 ms, followed by a black fixation cross  projected for 1,000 ms, and then, two collections of dots appeared at the left and right of the center of the screen  for additional 400 ms  composed of 168 randomly ordered trials were presented, for a total of 336 experimental trials , separated by a self-paced pause at the half of the whole session. The whole experiment lasted around 15–20 min.http://www.rstudio.com/) and Jamovi  softwares. Psychometric functions for each condition were generated by fitting Gaussian cumulative distribution functions to the data, and parameters were estimated with a parametric approach based on maximum likelihood method, using Quickpsy package for R  we calculated the coeffienct of variation  and the PSE of the psichometric functions for each illusion condition. Reaction times (RTs) for each illusions condition were also recorded. RTs’ data were logarithmically transformed and responses whose latencies fell outside of 1.5 times the interquartile range of the distribution were discarded . Two separated one-way repeated-measures analyses of variance (ANOVAs) were performed with the experimental condition  as within-subjects factor and with the mean PSE or the mean CoV as dependent variables. Furthermore, we performed a 4 × 4 repeated-measures ANOVA, with the absolute numerical distance between reference and test stimuli  and the experimental condition  as within-subjects factors and the mean RTs as dependent variable. The Greenhouse–Geisser epsilon (ε) correction for violation of sphericity was applied when needed and original F, df, and corrected p values were reported. Frequentist analyses were also accompanied by respective Bayesian analysis in the case of nonsignificant results.To investigate the effect of the illusions over perceived numerosity, we calculated the point of subjective equality (PSE) for each illusion condition as a function of the numerosity in test set—that is, the number of dots in test patterns required in order to be subjectively judjed as equal to the the reference patterns (12 items). The 50% of the chosen test patterns was set as threshold level. The 95% confidence intervals of individual PSEs were estimated running 200 bootstrap resampling of the data. Furthermore, as an index of the precision of the numerical discrimination and to confirm that the performance follows Weber’s law  = 55.1, ε = .89, p < .001, ηp2 = .45  and the Kanizsa condition (12.24 ± 0.39), t(198) = −2.79, p = .012, the baseline and the Ponzo condition (12.74 ± 0.41), t(198) = −8.99, p < .001, the baseline and the combined condition (12.92 ± 0.62), t(198) = −11.26, p < .001, as well as between the Kanizsa condition and the Ponzo condition, t(198) = −6.2, p < .001, between the Kanizsa condition and the combined condition, t(198) = −8.47, p < .001, and crucially, between the Ponzo condition and the combined Ponzo–Kanizsa condition, t(198) = −2.27, p = .024. No significant effect of the experimental illusion condition was found over the mean CoV, F = 1.06, p = .36, ηp2 = .016  showed a significant effect of the experimental condition, see Fig. . Post hosee Fig. . To furtF = 0.433, ε = .88, p = .70, ηp2 = .007; BF10 = .004  = 43.73, ε = .87, p < .001, ηp2 = .87. Trend analysis showed a significant linear decrease of RTs when numerical distance increases, t(198) = −10.11, p < .001. Lastly, no significant interaction between variables was found, F = 0.56, ε = .73, p = .778, ηp2 = .008. Results from Bayesian analysis suggest a strong evidence against including the interaction, BF10 = .0008 .Finally, the ANOVA on RTs showed no significant main effect of the illusion condition on RTs, In Experiment 2, participants performed the same number comparison task of Experiment 1, but in the combined condition the biases of the two illusions were modulated in a conflicting direction. That is, the Ponzo illusion triggered an overestimation bias, while the Kanizsa illusion an underestimation bias. If discrete information is processed independently from continuous features, when the two individual illusions are combined over the same stimulus in a conflicting direction, we should expect participants’ bias to be halfway as compared with the single illusory conditions, indicating that the two illusions compete against each other.Mage = 30.9 years, SD = 12.9 years, 49 females, 55 right-handed) was recruited for this study, which was also performed online due to COVID-19 restrictions.A new sample of 68 participants  = 36.3, p < .001, ηp2 = .35  = −2.69, p = .015, the baseline and the Ponzo condition (11.58 ± 0.61), t(201) = 6.74, p < .001, the baseline and the combined Ponzo–Kanizsa condition, (11.79 ± 0.55), t(201) = −4.47, p < .001, between the Kanizsa condition and the Ponzo condition, t(201) = 9.43, p < .001, the Kanizsa condition and the combined condition, t(201) = 7.17, p < .001, and, crucially, between the Ponzo condition and the combined Ponzo–Kanizsa condition, t(201) = −2.22, p = .025. As expected by the direct account of numerosity, the PSE of the combined condition was between the PSE of the illusion in isolations.Data were analyzed analogously to Experiment 1. The ANOVA on PSE showed a significant effect of the experimental condition,  see Fig . Post hoF = 0.83, ε = .85, p = .46, ηp2 = .012  = 0.28, ε = .90, p = .81, ηp2 = .004; BF10 = .004  = 14.14, ε = .86, p < .001, ηp2 = .17. A significant decreasing linear trend was also found over the absolute numerical distance, t(201) = −4.84, p < .001. Finally, no significant interaction between variables was found, F = 0.80, ε = .81, p = .58, ηp2 = .012. Results from Bayesian analysis suggest a very strong evidence against including the interaction, BF10 = .0015 .Furthermore, as in the previous experiment, no significant main effect of the experimental condition was found over RTs (7% of the data were discarded), In this study, across two different experiments, we document a clear role of visual segmentation mechanisms in discrete numerosity processing  illusion with the bias induced by the Ponzo illusion . Indeed, if participants actually ignored discrete numerosity, no difference should be found between the combined condition and the Ponzo condition in isolation, since in both conditions we have exactly the same continuous cues (as well as between the no-illusions condition and the Kanizsa condition). This strongly suggests that in the merged condition there is a combined effect of both information—namely, of discrete (manipulated by ICs) and continuous information (manipulated by the Ponzo illusion). Notably, neither of the two types of information dominated the other, and hence no factor was lost or ignored—they were simply combined (according to the direction of biases in each experiment).However, we also found a clear bias triggered by the Ponzo illusion when this illusion was presented in isolation. In line with previous studies using the Ebbinghaus illusion, we found that the Ponzo illusion led to an underestimation (Experiment 1) or an overestimation (Experiment 2) of test numerosity, depending on the context in which it was placed ESM 2(CSV 5 kb)ESM 3(CSV 8 kb)ESM 4(CSV 5 kb)ESM 5(DOCX 497 kb)


Artemisia spp. but, until now, has not been studied for its bioactive potential except antitrypanosomal activity. In this context, current study has reported the anti-adipogenic effect of DCC in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). DCC dose-dependently inhibited the lipid accumulation and expression of adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) in hBM-MSCs induced to undergo adipogenesis. To elucidate its action mechanism, the effect of DCC on Wnt/β-catenin and AMPK pathways was examined. Results showed that DCC treatment activated Wnt/β-catenin signaling pathway via AMPK evidenced by increased levels of AMPK phosphorylation and Wnt10b expression after DCC treatment. In addition, DCC treated adipo-induced hBM-MSCs exhibited significantly increased nuclear levels of β-catenin compared with diminished nuclear PPARγ levels. In conclusion, DCC was shown to be able to hinder adipogenesis by activating the β-catenin via AMPK, providing potential utilization of DCC as a nutraceutical against bone marrow adiposity.Increased bone marrow adiposity is widely observed in patients with obesity and osteoporosis and reported to have deleterious effects on bone formation. Dracunculin (DCC) is a coumarin isolated from  Worldwide prevalence of obesity is considered to be one of the main promoters for several common metabolic diseases such as diabetes, cardiovascular problems, and cancer . ReporteIn multipotent mesenchymal stromal cells (MSCs), increased adipocyte formation as a result of adipogenic differentiation promotes a set of bone disorders, osteoporosis being the most prevalent one ,5. AdipoArtemisia princeps, also known as Korean or Japanese mugwort, is a plant native to Asia and is reported to possess adipogenesis inhibitory properties in mouse 3T3-L1 pre-adipocytes among its other in vitro bioactivities such as antioxidant, antimicrobial, anti-inflammatory and antitumor [A. princeps as an active component. Eupatilin and jaceosidin flavonoids inhibited inflammation in mice [Artemisia species, tarragon (Artemisia dracunculus) being the first, which has given this compound its common name, dracunculin. Although several other coumarins were isolated from Artemisia species and their bioactivities have been reported, the literature lacks detailed information in terms of the health beneficial properties of dracunculin. Therefore, in this study, the potential anti-adipogenic effect of dracunculin (DCC) isolated from A. princeps has been evaluated. To the best of our knowledge, this is the first report that exhibits the effect of DCC on adipogenic differentiation.Natural bioactive substances have been gaining increasing attention due to their ability to regulate adipogenic differentiation. To date, several types of phytochemicals have been reported to possess adipogenesis inhibitory properties . Phenolintitumor ,10,11. S in mice  while 6- in mice  and loli in mice  inhibiteThe hBM-MSCs were treated with DCC A at the Any effect of DCC on the lipid accumulation of adipo-induced cells was observed by Oil Red O staining. As shown in Adipogenesis progresses via the activity of transcription factors such as PPARγ, C/EBP-α and SREBP1c . Therefo552 phosphorylated β-catenin levels. This phosphorylation of β-catenin via AMPK signaling stabilizes the transcription factor and consequently induces its nuclear translocation, which was previously noted as an adipogenesis suppressing mechanism [552 phosphorylated β-catenin levels diminished. Presence of 10 μM DCC reverted these adipogenesis induced changes: decreasing nuclear presence of PPARγ transcription factor while stimulating the β-catenin nuclear translocation.To elucidate the mechanism behind the effect of DCC on the PPARγ-related adipogenic differentiation, other signaling cascades that were shown to be in crosstalk and differentially regulating adipogenesis were analyzed. Reports showed that canonical Wnt/β-catenin signaling has an opposing relation with PPARγ where activation of Wnt/β-catenin subsequently downregulates PPARγ signaling and, therefore, adipogenesis is suppressed . In thisechanism . SubcellTo further support the reciprocal effect of DCC on the activation of Wnt/β-catenin and AMPK pathways, the activated AMPK protein levels were analyzed with or without the use of compound C, a known AMPK inhibitor. As shown in Next, the effect of DCC on AMPK activation was investigated. The hBM-MSCs were treated with compound C in the presence or absence of DCC (10 μM). Only compound C-treated hBM-MSCs showed significantly decreased phosphorylated AMPK levels after 6 and 12 h of treatment C. TreatiArtemisia spp. Although Artemisia spp. are known to possess anti-adipogenic properties and DCC is a common phytochemical, the literature lacks studies on the bioactive potential of DCC. Like other coumarins similar to DCC in terms of structure, DCC is expected to possess some bioactive properties. To the best of our knowledge, the only bioactive property reported for DCC is its antitrypanosomal activity [DCC is a coumarin derivative, also named as 7,8-Methylenedioxy-6-methoxycoumarin . It has activity . The curIncreased adipogenic differentiation of bone marrow cells is suggested to be one of the causes for obesity-related bone complications such as osteoporosis . FormatiThe adipogenesis of mesenchymal stromal cells is regulated by a web of different signaling cascades and transcription factors . The mai552 phosphorylated β-catenin levels were examined. When phosphorylated at Ser552 via AMPK, β-catenin is stabilized and accumulated in the nucleus, which consequently suppresses the PPARγ signaling [552 phosphorylated β-catenin levels in adipo-induced hBM-MSCs but did not affect the total β-catenin levels, indicating that DCC was able to activate β-catenin transcriptional activity through AMPK. This was further supported by the nuclear levels of phosphorylated β-catenin and PPARγ. Adipocytes expressed a high amount of PPARγ compared with a minimal presence of phosphorylated β-catenin in the nucleus. However, DCC treatment reverted the nuclear protein profile of adipo-induced hBM-MSCs. DCC-treated cells showed increased levels of nuclear phosphorylated β-catenin and diminished PPARγ. This supported the suggestion that DCC hinders the adipogenesis via activated β-catenin signaling.Another known regulator of adipogenesis is Wnt/β-catenin signaling, which downregulates PPARγ and C/EBPα cascade when activated . It has ignaling . DCC treA. princeps shares similarities with DCC in structure and was shown to inhibit adipogenic differentiation via similar pathways to further support the AMPK activating ability of DCC [AMPK is also known as a negative regulator for adipogenesis via different ways, which include suppression of MAPK activation on ERK1/2 and activation of β-catenin via phosphorylation. The stimulation of AMPK activation by DCC treatment was shown in this study. The presence of DCC significantly increased the AMPK phosphorylation, which was suppressed during adipogenesis. In addition, DCC was able to revert the effects of AMPK inhibitor compound C, further suggesting strong AMPK activation activity for DCC. Abiola et al.  suggestey of DCC .In conclusion, DCC inhibited the adipogenic differentiation and lipid accumulation of hBM-MSCs through activation of AMPK-mediated Wnt/β-catenin signaling in vitro. Future studies are expected to further examine its potential to be utilized as a natural bioactive agent against bone marrow adiposity.A. princeps and the chemical characterization of the DCC were carried out as reported [The isolation of DCC from reported . The NMRThe hBM-MSCs were differentiated into adipocytes as previously reported . Briefly2 incubator for 4 h. The wells were then aspirated and supplied with 100% DMSO. The absorbance values of the wells at 540 nm were measured on a MultiSkan GO microplate reader (Thermo Fisher Scientific). The cytotoxicity of DCC was measured as a relative percentage of cell viability compared to the untreated group.The cytotoxic concentrations of DCC were assessed by traditional MTT assay. The hBM-MSCs were grown in 96-well plates to approximately 80% confluency. Next, cells were treated with increasing concentrations of DCC in growth medium and incubated for 72 h. After incubation, growth medium was replaced with a fresh one containing 20 μL MTT (1 mg/mL) instead of DCC, and the cells were kept at 37 °C in a 5% COwt/v, dissolved in 3:2 isopropanol and distilled water). Staining was carried out by keeping the cells at room temperature for 1 h, after which the wells were aspirated again, washed with PBS and photographed using a microscope. Quantification of the Oil Red O stain bound by cellular lipid droplets was carried out via elution of the stain from cells through addition of 100% isopropanol. The wells were then measured for their absorbance at 500 nm on a MultiSkan GO microplate reader. The lipid accumulation was given as the stain absorbance percentage relative to the differentiated untreated control group.The Oil Red O staining was employed to demonstrate the cellular lipid accumulation as a marker of mature adipocyte. On day 12, the differentiated hBM-MSCs were washed with PBS and fixed on 6-well plates by addition on 10% formaldehyde for 1 h. After 1 h, the wells were aspirated and supplied with 0.5% Oil Red O staining solution  from each well. Reverse transcription was performed through the application of Cell Script All-in-One cDNA synthesis kit using a T100 thermocycler  with 42 °C for 60 min and 72 °C for 5 min settings. This was followed by standard PCR analysis consisting of 30 cycles of denaturing at 95 °C for 45 s, annealing at 60 °C for 1 min and extending at 72 °C for 45 s. The PCR reaction was carried out via Lune Universal PCR mix and following its instructions. The primers for the PCR analysis used were given in a previous report . Finallyg), and the supernatants were used for Western blot. Nuclear proteins were extracted from total cell lysates using a NE-PER nuclear extraction kit. The protein amounts of each well were assessed by BCA assay kit, and equal amounts of protein (20 μg) were loaded onto sodium dodecyl sulfate–polyacrylamide gel (4% stacking and 10% separating gels) for electrophoretic separation of the proteins. Next, the proteins were transferred to polyvinylidene fluoride membranes, which were then blocked with 5% skim milk in TBS-T buffer (v/v) for 1 h at room temperature. Blocked membranes were kept overnight at 4 °C with primary antibodies. Membranes were washed and hybridized with horseradish peroxidase-conjugated secondary antibodies. The hybridized protein bands were then visualized using enhanced chemiluminescence kit with a CAS-400SM imaging system.The Western blot was carried out to detect the protein levels of specific proteins in hBM-MSCs on day 12. The homogenized cellular total protein lysates were obtained using RIPA buffer and vigorous pipetting. The lysates were then centrifuged at 4 °C for 15 min  containing ProLong Gold Antifade reagent. Stained cell images were taken using an Olympus fluorescence microscope equipped with a digital camera, and the images were then processed with the Multi Gauge software.n = 3). The statistically significant difference between two groups was decided according to the results of one-way analysis of variance followed by Duncan’s multiple range post hoc test. SAS (v9.1) software was used for statistical analysis and calculation of p-values. The meaningful significance was determined at p < 0.05.The numerical data are given as mean of three separated results ± SD (


In summary, our study confirmed that SDF-1 and EX-4 combined application could enhance PDLSCs biological activity and promote PDLSCs osteogenic differentiation by regulating the metabolism, biosynthesis and immune-related signaling pathways.Stromal cell-derived factor-1 (SDF-1) and Exendin-4 (EX-4) play beneficial roles in promoting periodontal ligament stem cells (PDLSCs) osteogenic differentiation, while the detailed mechanism has not been clarified. In this study, we aimed to evaluate the biological mechanism of SDF-1 and EX-4 alone or synergistic application in regulating PDLSCs differentiation by RNA-sequencing (RNA-seq). A total of 110, 116 and 109 differentially expressed genes (DEGs) were generated in osteogenic medium induced PDLSCs treated by SDF-1, EX-4, and SDF-1+EX-4, respectively. The DEGs in SDF-1 group were enriched in signal transduction related signaling pathways; the DEGs in EX-4 group were enriched in metabolism and biosynthesis-related pathways; and the DEGs generated in SDF-1+EX-4 group were mainly enriched in RNA polymerase II transcription, cell differentiation, chromatin organization, protein phosphorylation pathways. Based on Venn analysis, a total of 37 specific DEGs were identified in SDF-1+EX-4 group, which were mainly enriched in negative regulation of autophagy and cellular component disassembly signaling pathways. Short time-series expression miner (STEM) analysis grouped all expressed genes of PDLSCs into 49 clusters according to the dynamic expression patterns and 25 genes, including NRSN2, CHD9, TUBA1A, distributed in 10 gene clusters in SDF-1+EX-4 treated PDLSCs were significantly up-regulated compared with the SDF-1 and EX-4 alone groups. The gene set enrichment analysis indicated that SDF-1 could amplify the role of EX-4 in regulating varied signaling pathways, such as type II diabetes mellitus and insulin signaling pathways; while EX-4 could aggravate the effect of SDF-1 on PDLSCs biological roles  Periodontal disease-induced tooth loss has become a global public health challenge that greatly affects people’s quality of life . MesenchAn increasing number of researchers have focused on the recruitment of endogenous PDLSCs to the injury site to enhance healing by harnessing the innate regenerative potential of the body . CytokinExendin-4 (EX-4), a full agonist of glucagon-like peptide-1 receptor (GLP-1R), has been widely used in the clinical treatment of type 2 diabetes mellitus (T2DM) . In addiHigh-throughput RNA sequencing (RNA-Seq) has been widely applied to analyze the whole transcriptomic changes of eukaryotes, which can provide progressively greater knowledge of both the quantitative and qualitative aspects of transcript biology . RNA-seqThis study was approved by the Medical Ethical Committee of the Stomatology School, Shandong University (NO. 20170801). Five healthy individuals without systemic diseases aged from 18–30 who underwent premolar extraction at the Department of Oral and Maxillofacial Surgery were recruited for this project. All individuals agreed to participate in the research project and signed the informed consent forms according to the Helsinki Declaration of 1975.2, and cells were trypsinized and passaged at a dilution ratio of 1:3 to expand the culture in 10% FBS medium upon the cell monolayer reached 80–90% confluence. Fourth passage cells were used in all experiments.The extracted teeth were stored in Dulbecco’s modified Eagle’s medium  with 5% antibiotics  and quickly transported from the clinic to the laboratory. Then, single PDLSCs were acquired as previously described in our previous study . Specifivia hierarchical indexing for spliced alignment of transcripts (HISAT) (v2.0.4)  by R package (v 3.2.5).PDLSCs from 5 different individuals were cultured in osteogenic medium (OM) and treated with SDF-1, EX-4 or SDF-1+EX-4 at 21 d, and normal PDLSCs treated with OM served as a negative control (NC). Totally, we collected 20 samples and RNA-seq was used to analyze the whole genome expression at LC Sciences through the Illumina X10 platform . Firstly, the total RNA were performed quality control based on previous study , and the(v2.0.4) . The map(v2.0.4) . FurtherVolcano analysis was used to identify the DEGs between each pair of groups . The up-q value <0.05 was used as the cut-off criterion for the selection of significant GO terms and KEGG pathways.Based on the DEGs generated by SDF-1, EX-4 and SDF-1+EX-4 compared with NC, the overrepresented GO categories and the significant KEGG pathways were identified . A q valhttp://jvenn.toulouse.inra.fr/app/example.html), and an intersection UpSet diagram based on the UpSet R package was drawn (http://metascape.org/gp/index.html#/main/step1)  .http://genemania.org/search) (STEM software (version 1.3.8)  was appl/search) .https://string-db.org/) and GeneMANIA database (http://genemania.org/search)  . Functio/search) .vs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs EX-4, SDF-1+EX-4 vs SDF-1, and EX-4 vs SDF-1 groups according to the clusterProfiler and enrichplot R package.GSEA is one of the functional class scoring analysis methods . To selevs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs EX-4, SDF-1+EX-4 vs SDF-1, EX-4 vs SDF-1  values of each gene at different time points post stimulation compared with NC were also calculated using DESeq2 R package. The thresh old value of —FC—>1 and FDR ≤ 0.05 was used to identify DEGs between two different groups, and the results indicated that 110, 116, 109, 125, 103 and 100 DEGs were generated in different comparison groups SDF-1 vs SDF-1 . Among avs SDF-1 .The GO  enrichment results showed that the DEGs generated by SDF-1 stimulation were mainly enriched in signal transduction regulation of transcription, cell differentiation, RNA polymerase II transcription, and multicellular organism development signaling pathways . The KEGvs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs EX-4, SDF-1+EX-4 vs SDF-1, and EX-4 vs SDF-1, we performed an overlapped analysis of the six compared DEG groups, and the results indicated that 34, 30, 37, 39, 35 and 28 DEGs were specially generated , and the results revealed 49 gene clusters among all DEGs generated by SDF-1, EX-4 and SDF-1+EX-4-treated PDLSCs . In all vs SDF-1, and SDF-1+EX-4 vs EX-4 were also centralized . The numbers of the DEGs are indicated at the top of every bar.Bar plot of differentially expressed genes (DEGs) of PDLSCs in SDF-1 Click here for additional data file.10.7717/peerj.12091/supp-2Supplemental Information 2vs NC (A), EX-4 vs NC (C), and SDF-1+EX-4 vs NC groups (E). KEGG enrichment analysis of the DEGs in the SDF-1 vs NC (B), EX-4 vs NC (D), and SDF-1+EX-4 vs NC groups (F). The size of the symbol represents the gene counts enriched in the signaling pathways. The color indicates the degree of statistical significance . The different color bars represent the different enrichment methods of the KEGG catalog .GO enrichment analysis of the DEGs in SDF-1 Click here for additional data file.10.7717/peerj.12091/supp-3Supplemental Information 3vs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs EX-4, SDF-1+EX-4 vs SDF-1, and EX-4 vs SDF-1 groups.UpSet plot analysis of the DEGs among the SDF-1 Click here for additional data file.10.7717/peerj.12091/supp-4Supplemental Information 4Short Time-series Expression Miner (STEM) analysis in PDLSCs cocultured with SDF-1, EX-4 and SDF-1+EX-4 .Click here for additional data file.10.7717/peerj.12091/supp-5Supplemental Information 5vs NC (A), EX-4 vs NC (B), SDF-1+EX-4 vs NC (C), SDF-1+EX-4 vs Ex-4 (D), SDF-1+EX-4 vs SDF-1 (E), and EX-4 vs SDF-1 (F).Network constructed through the STRING database based on the DEGs in SDF-1 Click here for additional data file.10.7717/peerj.12091/supp-6Supplemental Information 6vs NC (left panel) and the network based on the core genes generated by SDF-1+EX-4 (right). The genes in circles with white slash are the actual dynamic DEGs. The dynamic DEGs and predicted genes are interact based on physical interactions, coexpression, predications, colocalization, pathways, genetic interactions and shared protein domains.Network constructed through the GeneMANIA database based on the core DEGs in SDF-1+EX-4 Click here for additional data file.10.7717/peerj.12091/supp-7Supplemental Information 7vs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs EX-4, SDF-1+EX-4 vs SDF-1, and EX-4 vs SDF-1. Related to GO and KEGG functional enrichment analysis results based on DEGs in SDF-1 Click here for additional data file.10.7717/peerj.12091/supp-8Supplemental Information 8Related to Click here for additional data file.10.7717/peerj.12091/supp-9Supplemental Information 9Related to Click here for additional data file.10.7717/peerj.12091/supp-10Supplemental Information 10vs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs SDF-1, EX-4 vs SDF-1). Related to Functional enrichment analysis of the specific DEGs in each two compared groups  analysis in PDLSCs cocultured with SDF-1, EX-4 and SDF-1+EX-4. Related to Click here for additional data file.10.7717/peerj.12091/supp-12Supplemental Information 12Related to Click here for additional data file.10.7717/peerj.12091/supp-13Supplemental Information 13Related to Click here for additional data file.10.7717/peerj.12091/supp-14Supplemental Information 14Related to Click here for additional data file.10.7717/peerj.12091/supp-15Supplemental Information 15vs NC, EX-4 vs NC, SDF-1+EX-4 vs NC, SDF-1+EX-4 vs EX-4, and SDF-1+EX-4 vs SDF-1. Related to GSEA enrichment based on all genes in SDF-1 Click here for additional data file.


Chronic diseases (CDs) are noncommunicable illnesses with long-term symptoms accounting for ~70% of all deaths worldwide. For the diagnosis and prognosis of CDs, accurate biomarker detection is essential. Currently, the detection of CD-associated biomarkers is employed through complex platforms with certain limitations in their applicability and performance. There is hence unmet need to present innovative strategies that are applicable to the point-of-care (PoC) settings, and also, provide the precise detection of biomarkers. On the other hand, especially at PoC settings, microneedle (MN) technology, which comprises micron-size needles arranged on a miniature patch, has risen as a revolutionary approach in biosensing strategies, opening novel horizons to improve the existing PoC devices. Various MN-based platforms have been manufactured for distinctive purposes employing several techniques and materials. The development of MN-based biosensors for real-time monitoring of CD-associated biomarkers has garnered huge attention in recent years. Herein, we summarize basic concepts of MNs, including microfabrication techniques, design parameters, and their mechanism of action as a biosensing platform for CD diagnosis. Moreover, recent advances in the use of MNs for CD diagnosis are introduced and finally relevant clinical trials carried out using MNs as biosensing devices are highlighted. This review aims to address the potential use of MNs in CD diagnosis. From a medical perspective, chronic diseases (CDs) are noncommunicable illnesses with persistent symptoms requiring at least one year of continuous medical treatment . AccordiA valid measurement of biomarkers is vital for the diagnosis and prognosis of CDs ,5,6. CurBriefly, an MN-based sensing device consists of micron-sized arrays that are arranged on a miniature patch in a specific order. MNs are currently manufactured as solid, hollow, coated, and dissolving modes with a length and width of 150–1500 μm and 50–250 μm, respectively . MN-baseIn recent years, there has been enormous attention on the development of MN-based biosensors for the real-time monitoring of CDs  by puncturing the epithelial tissue  for the analysis of biomarkers . BiofluiThere is an urgent need for more reliable biosensing devices to provide timely feedback to patients diagnosed with CDs. Taken altogether, design parameters such as geometrical design, materials, fabrication, and coating methods of MNs should be further investigated to develop a more accurate MN-based sensing system for CD detection. Recent reviews broadly focus on the use of MNs for drug delivery purposes ,34,35. TMN fabrication techniques and material composition may vary depending on the needs and purpose of their applications . By usinMNs are major player in removing the limitations observed in conventional transdermal applications of the diagnostics or patient monitoring ,45. A nuMN-based platforms usually consist of two parts: (1) MN array that opens micropores in the skin through which biomarkers can be collected; (2) MN patch backing where the MNs are orientated on a surface. Patch backing can be fabricated together with the MN array or separately to further adhered to the arrays . Patch bThe MNs are divided into four main groups depending on their structure and usage strategies: (i) solid MNs that pre-treat the surface of the skin , (ii) coSolid MNs can be designed in different ways to form microchannels on the skin surface across the epidermis and dermis. These microchannels allow drugs or bioactive compounds to be delivered to the body through the dermis layer. These compounds are then transported to the desired places on the body by systemic circulation . Thanks ® MNs were produced using two-photon polymerization technique at different aspect ratios, and these MNs were able to penetrate inside porcine adipose tissue without cracking [In another study, Ormocercracking . Ceramiccracking . Even thcracking ,71,72. Lcracking ,74. Silicracking . Besidescracking . Additiocracking .Coated MNs are based on solid MNs, and they are formed by coating drug solutions, macromolecules, small molecules, vaccines, or micron-size particles on their structures ,77,78,79Conventional production techniques such as micromolding usually fail to change MN design quickly and easily due to the need for precise preparation processes and cumbersome master templates. Therefore, many different methods, such as dip coating, spray coating, and layer-by-layer manufacturing, have been tested in the production of coated MNs ,86,87,88Dissolving MNs mainly work on “the poke and release” principle. They have gained remarkable attention in recent years due to low-waste generation, affordable and facile production, self-administration, cold chain distribution, and higher patient compliance among the other MNs ,92. TheyDissolving MNs can be produced at low or room temperatures using mold-based techniques such as investment casting or hot embossing. Commonly, PVA , poly  method was developed , and basOn the other hand, micromachining methods are still not preferred because of their high production cost and time-consuming processes. However, Luzuriaga et al. have introduced a new method that they applied fused deposition modeling (3DP) and managed to quickly design and manufacture MNs at the desired density, length, and shape. Furthermore, by combining the 3DP method with the chemical etching method, the MN tips can be reduced to 1 mm in size .Another method used in the production of dissolving MNs is the atomized spray process, which is normally used in the production of different biopharmaceutical products. This method was used for the first time in the production of MNs, and it allowed the production of soluble MNs from sugar or polymers  . Since dHollow MNs have emerged as the conventional hypodermic needles decrease to micron sizes with the development of material technologies . CompareIn recent years, different types of MN-based biosensors including electrochemical, optical, magnetic, and paper-based have been studied in the literature ,144,145.Diabetes mellitus is an incurable disease caused by insulin deficiency in the body, resulting in hyperglycemia or hypoglycemia . Monitor2 nanoparticles, and a glucose-sensitive fluorophenylboronic acid-based matrix. These glucose-sensitive colloidal crystals were coated onto the polymerized MN surface through a secondary photocrosslinking method, which was mediated by residual double bonds on the MN. The resulting core-shell structure not only preserves the sensitivity of colloidal crystals, but also ensures that the MN has a suitable skin penetration feature. The MN patch reversibly converts glucose concentrations to color changes within 5 min that can be monitored by a naked eye.In another study, a biocompatible and invasive hollow MN platform integrated into a paper-based multiplex biosensor was designed to measure glucose and cholesterol levels in patients in multiple ways . After bThe demand for user-friendly and functional biosensor systems for the simultaneous detection of multiple blood metabolites has recently increased, especially for home-based health care and monitoring strategies. Based on this demand, a flexible MN electrode array-based biosensor and a portable multichannel electrochemical analyzer have been developed for the simultaneous detection of glucose, cholesterol, and uric acid . Here, t+) pair using the enzyme β-hydroxybutyrate dehydrogenase, and it was able to perform anodic detection of NADH , the MN-based system achieved to detect CEA biomarker in patients who have been diagnosed with breast cancer.Cancer has a growing impact on the world population and the health care system globally. Cancer is considered a CD by the WHO and Centers for Disease Control and Prevention since most of the cancer survivors require ongoing treatment for several years ,158. Eart cancer . Ag3PO4/−6 M for NO sensing. In addition, this system showed great stability for several days.Moreover, nitric oxide (NO) has been found to inhibit the progression of cancer by decreasing tumor growth, and it is another important biomarker to monitor cancer progression. In this manner, for the first time, the MN biosensor system was fabricated using polycaprolactone (PCL) and integrated into the endomicroscopic system . Poly  is a syndrome defined by changes in the kidney structure such as cysts, tumors, malformations, and atrophy. Furthermore, CKD has permanent effects such as edema in the kidney and changes in urine quality. These changes are usually described by increased levels of creatinine, cystatin C in serum, or urea nitrogen levels in blood . In a stParkinson’s disease (PD) is a chronic neurodegenerative disorder that results from a synaptic and neuronal loss due to the misfolding of α-synuclein (αSyn)―the major protein marker . Its cli−5 to 10−2 M with a near Nernstian slope of 57.9 mV and rapid stabilization (≈20 s)) than those of the porous graphene electrodes. They were able to measure potassium at normal physiological concentrations, spanning from 10–5 to 10–2 M. On the other hand, the porous graphene electrode-integrated MN showed instability during this operation and measurements.Electrolytes are pivotal elements for cell signaling , homeost+ and K+ in skin interstitial fluid was developed using micromilling machine [Moreover, skin interstitial fluids are rich sources of different types of electrolytes and their concentration is highly correlated with those in plasma. In a recent study, a MN-based potentiometric sensing system for continuous monitoring of Na machine . The holCurrently, approximately 62 clinical trials using MN technology have been reported on the ClinicalTrials.gov database . The usen = 8) and patients with Type 1 diabetes (n = 10), solid MN array-based biosensors were developed using polycarbonate MN arrays with a 1 mm thick base for monitoring glucose levels in real-time [According to the data obtained from clinicaltrials.gov, one of the clinical studies  was first posted on 15 February 2016, involving MN patches for continuous monitoring of glucose in diabetic patients. This study was conducted with fifteen children and adolescents (7 years to 18 years) with diabetes, enrolled between December 2017 and January 2018. The detection efficiency of the MN patch, which was made from biocompatible polymers and metals, was compared to the samples collected using a lancet and an intravenous catheter. MN patch was designed to collect glucose from interstitial fluid while lancet and intravenous catheter detected the glucose collected from capillary blood glucose and venous glucose, respectively. The samples were collected hourly from all three devices for four hours. According to the primary outcome, three methods showed similar detection efficiency. The pain level assessment by pain Visual Analog Scale (0 indicating no pain and 100 indicating the most extreme pain) showed that the MN patches, lancet and intravenous catheter caused pain levels of 11.2, 4.4, and 26.8, respectively. In another pilot clinical study with both healthy volunteers  with mild chronic plaque Psoriasis Vulgaris which is a skin disorder. The study completion date is reported to be in December 2019; however, no results have been posted yet on the outcome of this clinical trial. According to the Asthma and Allergy Foundation of America (AAFA), allergies are one of the most common CDs. MNs were tested in a clinical trial  on 20 participants (from 18 years to 65 years) with positive clinical history for inhalant allergy caused by birch pollen. The objective of this study was to determine allergic skin activity. Each patient was treated with three skin preparation techniques: (1) MNs, (2) Prick Lancet, and (3) tape stripping (Tesafilm). Unfortunately, no result is available on the results of this clinical trial since the last update in 2012.On the other hand, there are some recently completed and/orongoing clinical studies under this topic. For instance, a clinical trial  using an MN device was conducted in March 2019 to collect and analyze samples for transcriptomics profiling from patients (Stating our personal views in this context, real-time monitoring of CDs still remains a challenge. Essentially, the development of effective biosensing devices is crucial to overcome these limitations associated with the current PoC platforms. Out of different approaches, MN-based platforms have been proven to significantly improve the detection efficiency. Recently, a great variety of MN-based biosensing platforms have been introduced. Currently, MN technologies still face certain obstacles. One field that still needs attention is regarding the efficacy of in vivo sampling with MN-based biosensors. Unfortunately, to date, only a few clinical trials involving such sensors have been reported. Certain strategies need to be followed to accelerate the translation of MN-based platforms from preclinical development into clinical trials.One of the strategies is to propose easier and more affordable microfabrication techniques using more accessible materials to manufacture MNs. Advancements in ultra-rapid prototyping techniques would allow high-throughput manufacturing of MNs. For instance, one of the promising manufacturing technologies would be 3D printing. Recent breakthroughs in engineering inspired by the progress of photopolymerization techniques using a variety of material have made the manufacturing of such MNs with desired structures possible . HoweverIn a nutshell, the MN-based biosensor would be integrated both in the process of acquiring time-sensitive and clinically valuable information through sensing and/or in long-term wearable monitoring systems. Consequently, the advances in MN-based biosensors for medical applications for detecting CDs would contribute to global healthcare, as well as potentially reducing healthcare disparities, regardless of the socio-economic group, with advancements in large-scale and inexpensive manufacturing of these platforms. In due course, it is expected that new advances in MN technology would accelerate to have the complete control of disease progression.


The monomers of fatty acids, the low abundant ω, mid-chain-diOH fatty acids, and 2-hydroxy fatty acids increased remarkably under low temperature. The cuticular waxes were dominated by fatty acids (> C19), n-alkanes, and triterpenoids; and the fatty acids and aldehydes were shifted to increase accompanied by the chilling injury. Furthermore, RNA-seq highlighted 111 cuticle-related genes involved in fatty acid elongation, biosynthesis of very-long-chain (VLC) aliphatics, triterpenoids, and cutin monomers, and lipid-transfer proteins were significantly differentially regulated by low temperature in banana. Results obtained indicate that the cuticle covering on the fruit surface was also involved to respond to the chilling injury of banana fruit after harvest. These findings provide useful insights to link the cuticle on the basis of morphology, chemical composition changes, and their biosynthesis regulations in response to the thermal stress of fruit during storage.The plant cuticle covers almost all the outermost surface of aerial plant organs, which play a primary function in limiting water loss and responding to the environmental interactions. Banana fruit is susceptible to thermal changes with chilling injury below 13°C and green ripening over 25°C. Herein, the changes of surface morphology, chemical compositions of cuticle, and the relative expression of cuticle biosynthesis genes in banana fruit under low-temperature storage were investigated. Banana fruit exhibited chilling injury rapidly with browned peel appearance stored at 4°C for 6 days. The surface altered apparently from the clear plateau with micro-crystals to smooth appearance. As compared to normal ones, the overall coverage of the main cuticle pattern of waxes and cutin monomers increased about 22% and 35%, respectively, in browned banana stored under low temperature at 6 days. Fatty acids (C Banana is one of the most populated horticultural crops planted widely in the tropical and subtropical regions including south China. Banana contains abundant bioactive phytochemicals such as carbohydrates, vitamins, minerals, etc., being one of the most consumed fruit worldwide . Low-tem16 and C18 fatty acid monomers polymerized into the framework that harbors the various waxes  and triterpenoids in desert plant leaves  at physiological mature stage with green were harvested from an orchard in Guangzhou, Guangdong Province, P.R. China . Harvested fruits were packed with polypropylene plastic bags (film thickness: 32 μm) and immediately transported to the laboratory within 2 h. Fruits were selected for uniformity of shape, color, and size, and rinsed the surface gently. Then fruits were placed into two groups randomly and packed in polyethylene bags  and stored at 4 and 25°C, respectively. Six bags in total of 18 fingers from each treatment were randomly selected at 0, 2, 4, and 6 days during storage, and the chilling index and hue angle were evaluated. To isolate cuticular membranes, chemical reagents of analytical grade were prepared as previously described / × 100.h° = tan–1(b*/a*)].The peel color degree was determined using a Minolta Chroma Meter CR-400 . Nine fruit fingers from each treatment at each stored stage and three points around the middle of each fruit were measured. Color was recorded as parameters of CIE L*, a*, and b* scales. In which, L indicates the lightness or darkness, a* indicates green to red, and b* donates blue to yellow. The overall color index was indicated by L* or general value of hue angle [The surface characteristics of each organ of the two kinds of vegetables were observed using scanning electron microscopy (SEM). Briefly, small pieces (3 mm × 3 mm) of the peel tissues were picked and fixed in 2.5% glutaraldehyde in 0.2 M sodium phosphate buffer, pH 7.2–7.4 at 4°C overnight. The fixed samples were vacuum-infiltrated in the material on ice for 1 h. The samples were washed using sodium phosphate buffer and then dehydrated in a graded alcohol series with 30, 50, 75, 90, and 100% three times. After the samples were dried completely, the small pieces were mounted on aluminum stubs using a conductive double-sided adhesive tape prior to observation. The dried samples were coated with gold:palladium (60:40) at 30 mA using a LEICA EM ACE600  sputter coater, depositing an alloy of approximately 10 nm thickness. The characteristics of the sample surfaces were examined under a field emission SEM  at 15 kV accelerating voltage.The cuticles of banana fruit were isolated enzymatically following the protocol described by n-tetracosane  as an internal standard was added to help detect the accumulation of cuticular waxes. Gentle stream of nitrogen gas was used to dry the extracts for further analysis.The cuticular waxes and cutin monomers of banana fruit were extracted using the isolated cuticular membranes following the reported procedure previously . The dry3-methanol, 10%, ∼1.3 M) overnight at 70°C. Then, n-dotriacontane  was added as an internal standard. Saturated aqueous sodium chloride solution and chloroform were added subsequently to extract the cutin monomers. The organic phase containing cutin monomers was collected and evaporated to dryness under a gentle stream of nitrogen gas for further analysis.The above matrix membrane after removing waxes was subsequently depolymerized in boron trifluoride with methanol (BFbis (trimethylsilyl)trifluoroacetamide  for 30 min at 70°C. To quantify wax and cutin monomer components, the extracts were analyzed using a capillary gas chromatography instrument equipped with a flame ionization detector , and on-column injection with a capillary column . To separate cuticular wax compounds, 10 μL samples were injected at 50°C; after 2 min at 50°C, the temperature was raised to 200°C at 40°C min–1, held for 2 min at 200°C, and then raised to 320°C at 3°C min–1 and held for 30 min at 320°C. For separation of the cutin monomers, 10 μl samples were injected at 50°C; following 1 min at 50°C, the temperature was raised to 150°C at 10°C min–1, held for 2 min at 150°C, and then raised to 320°C at 3°C min–1 and held for 30 min at 320°C. The area under the peaks was compared with that of the internal standards to obtain the quantity of cuticular wax and cutin monomer components.Prior to the analysis of the chemical composition of cuticular waxes and cutin monomers, the dry extracts prepared in the aforementioned manner were derivatized with pyridine  and N,O-m/z 50–750, MSD 5975; Agilent Technologies, Santa Clara, CA, United States). Single compounds were identified based on their electron ionization mass spectra using authentic standards, the Wiley 10th/NIST 2014 mass spectral library , or by interpretation of the spectra according to the retention times and/or by comparison with data from the literature  and Agilent 2100 Bioanalyzer . Then the cDNA was synthesized by mixing with DNA polymerase I and RNase H. The samples were amplified and sequenced on Illumina HiSeq™ 4000 by Gene Denovo Co. . The fine data were mapped to the reference genome using published banana data. The gene expression abundance was represented by an RPKM value. Differentially expressed genes (DEGs) were identified by the DESeq22, which indicated the change of false discovery rate genes over twofold with statistical significance . DEGs in the pointed pathways were analyzed in detail.Three biological replicates were performed for each sample. Total RNA was extracted and removed the contaminations with DNase I. Then, the purity and concentration of the RNA were checked by multifunction microwell plate detector  template kit , and their concentrations were determined using a multifunction microwell plate detector . Relative expression levels of the sequenced DEGs related to fatty acid elongation  and wax biosynthesis (MaCER1 and MaFAR) were carried out by qRT-PCR. The reaction system was SYBR Premix Ex Taq Kit  where PCR conditions for the primers were 95°C for 30 s, 95°C for 5 s, and 60°C for 34 s, 40 cycles. The Primer Premier 6.0 software  was used to design the primers  in an ABI7500 Real-Time PCR System . Three independent biological replicates were used in the analysis.The total RNA of banana peel tissues using a HiPure Plant RNA Mini Kit  and cleaned with DNase I . The DNA-free RNA was used as the template for reverse-transcription PCR (RT-PCR). The first-strand cDNA template of each gene was synthesized using PrimeScript primers . The qRTStatistical analyses were performed using the IBM SPSS  and SigmaPlot 12.5 . Comparison analyses were performed by one-way ANOVA, and significant differences were analyzed at a level of 0.05. SigmaPlot 12.5 was used to elaborate the graphs shown in the figures.Banana fruit exhibited an obvious browning appearance as the chilling injury symptom with an index over 80% of surface-browned at 6 days under 4°C after harvest . The hueThe SEM observation showed the morphological changes of banana peel surface stored at 6 days under different temperatures. The epidermis of banana fruit consists of small square-, rectangular-, and gourd-shaped epidermal cells. These epidermal cells were outward forming numerous plateau ridges . Similar–2 in green mature fruit stored at 25°C, which was shifted to increase significantly (p < 0.05) as 31.2 μg cm–2 by chilling injury after 6 days  under both temperature storage. As a result, the ratio of aliphatics over cyclics was shifted from 2.6 to 3.3  were detected within a fairly broad range of carbon chain lengths. Fatty acids as the most abundant aliphatic pattern ranged from C20 to C30 with most abundant of C22 and C24, which raised significantly under low-temperature storage at 6 days compared to that stored under 25°C  and primary alcohols (C20 to C32) accumulated with prominent of C28 and C30, which increased for C30 under low temperature  downregulation and 7,287 DEGs (44%) upregulation in this study. The Gene Ontology (GO) annotation indicated that 7,576 DEGs  were identified as “biological process” with 18 functional groups; 4,834 DEGs (30.5%) were annotated as “cellular component” with 14 functional groups; and 3,464 DEGs (21.8%) were involved in “molecular function” with 10 functional groups . The Kyon-alkane pathways, cyclic waxes, cutin biosynthesis, and lipid-transfer proteins (As cuticular constituents were mostly constituted by lipids, a total of 392 DEGs in 15 different pathways involved in lipid metabolism were detected. Fatty acid elongation, ether lipid metabolism, biosynthesis of unsaturated fatty acids, etc., were enriched markedly (rich factor > 0.5, proteins .MaLCAS1/2/9, MaKCS1/2/3/4/11, MaKCR1, MaECR, and MaCER26/26L (MaCER1/3/7, MaFAR1/4, MaWSD1, MaDGAT1/2, MaSQS1/2, and Masqe1/3 (MaCYP74A, MaCYP74A2, MaCYP86A1/2, MaCYP86B1, MaHHT1, MaHHT, MaPXG4, MaDCR1/2/3, MaGPAT1/3/4/5/6/7, and MaAGPAT6 were differentially regulated (MaLTP1/2, MaLTPG1/2, and MaABCG11, which were potentially related to the transportation of cutin monomers and waxes were detected (Among these cuticle-related genes, 23 DEGs were involved in fatty acid elongation including ER26/26L . BesidesMasqe1/3 . About 2egulated . And 12 detected .16 and C18 fatty acids. The chilling conditions could shift the ratio of saturated and unsaturated fatty acids in the plasma membrane and induce lipid peroxidation (The present study focused on characterizing the response of cuticle to the chilling injury in banana fruit under low temperature using room temperature as control. Banana is a typical horticultural crop that is sensitive to temperature changes during growing and postharvest storage. The chilling injury with browning of the peel tissues and discoloration, and informal firmness in banana fruit occurred after harvest. Low temperature disturbed the distribution of oxidation-related enzymes and the polyphenolics in plant cells, inducing the oxidation of polyphenolics in peel tissues . In addixidation .Similarly, besides the membrane lipids, the changes in cuticle, which are prominently lipid compounds, might also be involved in response to the temperature stress in banana fruit. The cuticle strength and stiffness showed largely to be affected by temperature changes . The hig30 (28. Accordingly, the accumulation of C22 and C24 as relatively short carbon chains in the VLC fatty acids in waxes increased in banana under low temperature (In banana fruit, the surface microstructure was apparently disturbed by low temperature after 6 days following the occurrence of chilling injury . The str30 . Compareperature , 5.n-alkanes with C29 and C31, or stimulated the increase of VLC alkyl esters in plant leaf or fruit cuticles (n-alkane and alcohol forming pathways. It was consistent with the relatively stable coverage of alcohols and n-alkanes, which only increased slightly for aldehydes with C30 and n-alkanes with C25 during storage (MaLACS, MaKCS, MaKCR, MaECR, MaCER1, and MaFAR was selected to further perform the qPCR analysis. The relative expression level of these genes was stimulated to increase by the low temperature in 4 days, but declined rapidly following the chilling injury (SQS and SQE members were as the DEGs involved in biosynthesis of triterpenoids (The cuticular waxes are stimulated to accumulate by the environmental stresses, i.e., the drought stress improved the accumulation of VLC cuticles . In bana storage , 5, 8B. g injury . In addig injury . In banag injury . Numerourpenoids . In contrpenoids , declinep-coumaric acid and their derivatives, which were common in plant cuticles, were also detected in banana fruit. These small molecules of phenolics are thought to be mainly embedded in the intracuticular layer together with cutin polymers (16 and C18 fatty acids, the biosynthesis of cutin monomers regulated by CYP74 and CYP86 members, HHT, DCR, and GPATs exhibited homeostasis status with an equal number of 13 genes for downregulation and upregulation, respectively (The cutin monomers polymerized as matrix is another main part of cuticle besides waxes. Genetic monitoring of the cutin deficiency induced the susceptible infection of microbial in tomato fruit . Howeverpolymers . It mighectively . These r16 and C18 fatty acids, were detected in banana fruit cuticle.In addition, 12 lipid-transfer proteins involved in cutin monomer and wax secretion were also detected to be differentially regulated in banana cuticle under low temperatures . General16 and C18 as the monomers in cutin, and C22 and C24 fatty acids and cyclic sterols and triterpenoids in wax mixtures were the most changed cuticular components. To respond the chilling injury, a series of cuticle-related genes, i.e., MaLACS, MaKCS, MaKCR, MaCER26, MaCER1/3, and MaFAR in cuticular wax biosynthesis and MaCYP86, MaCYP86A1/4, MaPXG4, MaHTH, and MaGPAT6 involved in cutin monomer accumulation, were differentially expressed in banana fruit (This study in detail reported the changes of appearance morphology, response of chemical compositions, and their biosynthesis regulation genes in banana cuticle under low-temperature storage. Our study demonstrated that the appearance of banana fruit was altered apparently into flat paralleled with changes of cuticular wax and cutin monomers in cuticle stimulated by the chilling injury. Fatty acids of Cna fruit . In addiThe original contributions presented in the study are included in the article/HH designed and prepared the manuscript, performed most of the experiments, and prepared the draft of the manuscript. LW, DQ, and NZ contributed to part of the experiments and data analyses. FB took part in revising the manuscript and funding acquisition. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.