Network pharmacology revealed that fucosterol targets signaling molecules, receptors, enzymes, transporters, transcription factors, cytoskeletal, and various other proteins of cellular pathways, including tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), neurotrophin, and toll-like receptor (TLR) signaling, which are intimately associated with neuronal survival, immune response, and inflammation. Moreover, the molecular simulation study further verified that fucosterol exhibited a significant binding affinity to some of the vital targets, including liver X-receptor-beta (LXR-β), glucocorticoid receptor (GR), tropomyosin receptor kinase B (TrkB), toll-like receptor 2/4 (TLR2/4), and β-secretase (BACE1), which are the crucial regulators of molecular and cellular processes associated with NDD. Together, the present system pharmacology and in silico findings demonstrate that fucosterol might play a significant role in modulating NDD-pathobiology, supporting its therapeutic application for the prevention and treatment of NDD.Fucosterol is an algae-derived unique phytosterol having several medicinal properties, including antioxidant, anti-inflammatory, anticholinesterase, neuroprotective, and so on. Accumulated evidence suggests a therapeutic promise of fucosterol in neurodegeneration; however, the in-depth pharmacological mechanism of its neuroprotection is poorly understood. Here, we employed system pharmacology and  Neurodegenerative disorders (NDD), such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) are the major causes of dementia worldwide. Although the exact mechanism is unclear, inflammation and oxidative stress might play pivotal roles in the pathogenesis of NDD . The celPhytosterols are important molecules not only for their structural and signaling functions in the host plants  but alsoin silico analysis offers an efficient computation tool that can better explain how a bioactive molecule interacts with the signal molecules of various cellular pathways and contribute in the therapy of multifactorial disease like NDD. Here, we employed integrated system pharmacology and in silico analysis to explain the pharmacological mechanism of fucosterol against NDD. First, through multiple bioinformatics tools, we demonstrate that fucosterol highly interacted with signaling pathways related to the development and survival of neuron, inflammation and immune response. Next, we employed in silico analysis to verify the biophysical interaction of fucosterol with some potential targets. The present study elucidates the underlying mechanism of molecular neuropharmacology of fucosterol and establishes a basis in favor of its therapeutic application against neurodegeneration.The system biology approach combined with A diagrammatic overview displaying the different steps of network pharmacology was illustrated in A total of 72 potential targets of fucosterol were retrieved from Pathway Assembly from Literature Mining-an Information Search Tool (PALM-IST) database  and valiBDNF, APP, APOE, AKT1, PPARG, NFKB1, NFE2L2 (Nrf2), CYSC, MMP9, MAPK1, SOD1, JUN, FOS, CAT, TNF, IL6, IL1B, NOS2, CASP3, CASP9, and HMOX1 are the dominant hub genes that participate in the major cellular pathways.F–T–T network demonstrated the interaction between fucosterol and its targets as well among the targets and identified most interacted protein targets in the network A. This nBDNF, BCL2, BAX, ATF2, APP, TNF, APOE, TGFB1, AKT1, SOD1, AChE, ACE, PPARG (PPAR-γ), PIK3CA, ABCA1, NFKB1, NFE2L2 (Nrf2), MMP9, MAPK1, IL6, IL1B, HMOX1, GRIN2A (GluN2A), CAT, CASP3, and CASP9, each was connected with NDD, indicating the involvement of these targets or others to their upstream signaling as a potential drug-target for fucosterol in the NDD treatment. Moreover, there are some other potential targets, including NTRK2 (TrkB), NR1H2 (LXR-β), NR3C1 glucocorticoid receptor, (GR), and β-secretase, that are closely associated with pathways and cellular processes in NDD pathology.F–T–NDD network presented the interaction of fucosterol with those targets that are associated with NDD. A total of 56, 41, and 34 targets, respectively, were associated with AD, PD, and HD B. Among Top 10 highly enriched GO terms under cellular component (CC), biological process (BP), and molecular function (MF) were displayed A–C. The p-value < 0.01) in the “signal transduction” module  pathway annotation. A total of 15 signaling pathways were enriched , synaptic plasticity , inflammation , and apoptosis  (COX2) and apoptosis-related proteins  were targeted by fucosterol (SOD1 and GPX1), neuronal growth and survival , inflammation , and apoptosis  were the targets of fucosterol , cholesterol homeostasis and Aβ-clearance , neuronal growth and survival  A. Among costerol B. On thecosterol C.We employed molecular docking analysis to validate the interaction patterns and efficiency of fucosterol to some of the vital target proteins, which are crucial regulators of several NDD-related molecular and cellular processes. Here we selected LXR-β, GR, TrkB, TLR2/4, BACE1, and AChE depending on their crucial roles in the inflammation, oxidative stress, apoptosis, and other risk factors associated with the pathobiology of NDD. Molecular docking followed by binding energy analysis showed that fucosterol showed the highest binding affinity to LXR-β, followed by GR and TrkB with binding energies of −80.37, −49.53, and −34.06 kcal/mol , respectAs an initial consideration, binding and interaction patterns revealed from molecular docking simulation were assessed. The result demonstrated that fucosterol formed significant interactions with these receptors following hydrophobic interactions. As shown in The findings from molecular docking studies were further validated by 50 ns additional molecular dynamics simulations. Initially, the root means standard deviation (RMSD) of both protein and ligand from individual complexes was calculated by comparing with their initial position and represented in Interestingly, fucosterol maintained contacts with the active site residues, including Ile353, Leu345, Leu442, Phe268, Trp457, Ala275, Phe340, and Phe243 residues over the 80% of total simulation time, which were consistent with molecular docking simulation. In addition, fucosterol also showed maximum interactions with Arg319 and Glu281 residues during the simulation. Consequently, Met604, Leu608, and Phe623 residues in GR showed maximum contacts with fucosterol, while TrkB maintained hydrophobic interactions through the Ile323, Leu324, and Tyr329 residues. Furthermore, hydrogen bond occupancy showed that the hydroxyl group of fucosterol maintained hydrogen bond with Glu281 of LXR-β during the 10.74% of total simulation time but not with Asn239, which concluded that the conformational drift that exerted by fucosterol at the initial stage of simulation, changed the binding orientations, which might be more favorable and stronger than the complex predicted from docking simulation.Physiologically, the activation of human LXR by either sterol and nonsterol agonists is usually exerted by a perpendicular histidine-tryptophan switch in the ligand binding domain, which turns the receptor into its active conformation, where the sterol guided activation is mediated by Trp457 . Having The sifting of interaction pattern from Arg319 in molecular docking to Glu281 in molecular dynamics simulation suggests that fucosterol might induce conformational changes and thus activates LXR-β. In the case of GR, fucosterol showed a similar hydrogen bonding profile, where the compound maintained 8.94% of hydrogen bonding occupancy with Gln570; however, the hydrogen bonding pattern remained stable for TrkB, where Ile323 made 9.55% of H-bonding during the simulation. Together, it can be concluded that hydrophobic interactions play a significant role in the binding of fucosterol to LXR-β, GR and TrkB.Several earlier reports highlighted that the fifth subdomain at the extracellular part of the TrkB receptor plays major role in the BDNF mediated TrkB interactions ,40,41,42Here, we unraveled the molecular mechanism of neuropharmacological action of fucosterol against neurodegenerative disorders using integrated system pharmacology and molecular simulation approach. Initially, we characterized the pharmacokinetic behaviors of fucosterol, which supports its drug-like properties and accessibility to the brain tissue. Moreover, referring to its cholesterol-like chemistry, fucosterol could pass through the cell membrane to reach directly to various intracellular targets. The network pharmacology data indicated that fucosterol showed a close association with the target proteins of many crucial pathways at the molecular and cellular levels. Gene ontology-based bioinformatics analysis identified several enriched biological processes, including inflammatory response, apoptotic process, transcription regulation, aging, NF-κB activity, and cellular response to ROS, which are highly linked with the pathobiology of NDD. KEGG pathway analysis further demonstrates that fucosterol targeted proteins are the critical components of these highly enriched molecular and cellular pathways.in silico data showing a significant binding affinity of fucosterol to LXR-β suggest the involvement of a similar mechanism. Moreover, the glucocorticoid receptor (GR), upon ligand binding, exerts its potent anti-inflammatory actions through repressing NF-κB signaling [In the pathway analysis, inflammation-related pathways, including TNF, HIF-1, NF-κB, and VEGF signaling, are particularly enriched, indicating that the anti-inflammatory action of fucosterol could play a significant role in the treatment of NDD. Being a ubiquitous transcription factor that plays a crucial role in amplifying inflammatory and immune responses , NF-κB pignaling . The preApoE, ABCA1, ABCG1, and SREBF1, the key genes in reverse cholesterol transport [LXR target genes, such as ABCA1, ABCG1, and ApoE [Given the key role of cholesterol in brain physiology and function, disturbances in cholesterol homeostasis provoked inflammation and oxidative stress, and thus, have been associated with the onset of major NDDs . LXRs, pransport . The actransport ,52. Hoanand ApoE . A similand ApoE . These fAnother enriched signaling pathway is the PI3K/Akt pathway, which, along with MAPK signaling pathway, regulates cellular growth and survival. This pathway has multiple downstream effectors, including those involved in cell survival , which suggests that this compound might function as BDNF-mimetic. Moreover, PI3K/Akt signaling pathway downstream to the neurotrophin signaling pathway was one of the enriched pathways in our study, indicating that these pathways might be employed in the pharmacological actions of fucosterol against NDD. In addition, Oh and colleagues reported that fucosterol protects against Aβ1-42-induced cytotoxicity through activating TrkB-mediated ERK1/2 signaling in primary hippocampal neurons, a TrkB-dependent neuroprotective effect, which was reversed by a selective TrkB inhibitor, cyclotraxin B, indicating its physiological interaction with TrkB in neuronal cells . These iathology ,55. A prathology  and our 2+ signaling, and thus plays a significant role in synaptic plasticity, which constitutes the biochemical basis of memory and cognition. The agonistic behavior of fucosterol to GluN2A could, therefore, help improve cognition deficits in AD patients. On the other hand, AChE catalyzes the breakdown reaction of acetylcholine at the cholinergic synapse, regulating synaptic transmission. However, AD pathology is accompanied by the cholinergic deficit, which could be compensated by the AChE inhibitor. Previous studies showing the inhibitory activity of fucosterol against AChE [Moreover, some vital synaptic proteins, including GluN2A and AChE were connected to fucosterol. GluN2A at the glutamatergic synapse mediates Canst AChE ,28 and oin silico analysis with this protein target. Molecular docking analysis demonstrates that fucosterol interacted with both TLR2 and TLR4, suggesting that this compound could improve inflammation-induced NDD pathology through TLR-mediated immune response. In addition to their ability to activate the expression of genes linked to lipid metabolism, LXRs also antagonize inflammatory gene expression triggered by TLR activation [In the KEGG pathway analysis, there was an enrichment of several immune-related pathways that are deregulated and compromised in NDD. In particular, several protein molecules of the TLR signaling pathway were targeted by fucosterol. TLRs play a critical role in innate immunity by recognizing pathogen-associated molecular patterns . Deregultivation .in silico analysis also revealed that fucosterol interacted with β-secretase, suggesting its potent anti-amyloidogenic activity.β-Secretase cleaves amyloid precursor protein leading to the production of Aβ in the brain. Aberrant β-secretase activity is believed to be implicated in the pathogenesis of AD. Thus, targeting β-secretase is a viable therapeutic approach for AD. However, evidence suggests that the complete abolishment of β-secretase may be associated with specific behavioral and physiological alterations . TherefoPharmacokinetic parameters including absorption, distribution, metabolism, and excretion/transport (ADME/T) of fucosterol were analyzed through QikProp . QikProp is an efficient ADME/T prediction tool that forecasts whether the selected compound would exhibit satisfactory ADME/T performances during clinical trials.http://www.uniprot.org/) [The information on fucosterol targets was retrieved primarily from PALM-IST database. We verified every target protein through the PubMed link. We excluded those targets that have no direct interaction with fucosterol and included additional targets while scanning PubMed database. The protein information, including protein name, gene ID, and host organism were verified through UniProt (ot.org/) .A protein–protein interaction (PPI) network among the targets was built using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), an online tool for functional protein association networks. The fucosterol-target network and PPI network were merged through Cytoscape v3.7.1  . This neThe lists of AD, PD, and HD-related genes were retrieved from DisGeNET database v6.0  , a databhttps://www.networkanalyst.ca/). GO terms with a p-value of < 0.05 were considered significant. The functional enrichment analysis of GO for the biological process, molecular function, and cellular components was performed and presented in bar graphs using Network Analyst  . Pathways highlighting fucosterol targets were retrieved through the KEGG pathway mapper [https://www.genome.jp/kegg/tool/map_pathway2.html).Gene functional annotation for KEGG pathways was retrieved from the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. First, gene counts for each pathway in the given gene set were calculated. Then, significantly enriched pathways in the given gene set compared to the genome background are defined by a hypergeometric test. Taking database  , GR (PDB ID: 6DXK), TrkB (PDB ID: 1HCF), TLR2 (PDB ID: 6NIG), TLR4 (PDB ID: 5IJC), BACE1 (PDB ID: 5HDZ), and AChE (PDB ID: 3QT0) were retrieved from the protein data bank  and prepAfter that, the docked complexes were subjected to molecular dynamics simulations using Yet Another Scientific Artificial Reality Application (YASARA) v.16.9.23 (YASARA Biosciences GmbH) Dynamics software. Before the simulation, all structures were cleaned and subjected to the optimization of hydrogen bonding network. For each simulation system, a cubic simulation cell with periodic boundary condition was generated and then all atoms were parameterized with the Assisted Model Building with Energy Refinement (AMBER14)  force fiin silico findings demonstrate that fucosterol interacted with the proteins of major molecular and cellular pathways that are involved in neuronal growth and survival, neuroinflammation, and immune response, and thus could help modulate and improve pathobiology of neurodegeneration. Our system biology approach to unravel the neuropharmacological action mechanism of fucosterol further offers a platform to explain the complex pharmacological mechanism of a bioactive molecule against NDDs in a multitarget approach. However, further experimental validation by cellular and animal studies is warranted before its recommendation for clinical application.The network pharmacology and


Salvia miltiorrhiza (S. miltiorrhiza) is an important Chinese herb that is derived from the perennial plant of Lamiaceae, which has been used to treat neurasthenic insomnia and cardiovascular disease. We produced a mutant S. miltiorrhiza (MT), from breeding experiments, that possessed a large taproot, reduced lateral roots, and defective flowering. We performed transcriptome profiling of wild type (WT) and MT S. miltiorrhiza using second-generation Illumina sequencing to identify differentially expressed genes (DEGs) that could account for these phenotypical differences. Of the DEGs identified, we investigated the role of SmGASA4, the expression of which was down-regulated in MT plants. SmGASA4 was introduced into Arobidopsis and S. militiorrhiza under the control of a CaMV35S promoter to verify its influence on abiotic stress and S. miltiorrhiza secondary metabolism biosynthesis. SmGASA4 was found to promote flower and root development in Arobidopsis. SmGASA4 was also found to be positively regulated by Gibberellin (GA) and significantly enhanced plant resistance to salt, drought, and paclobutrazol (PBZ) stress. SmGASA4 also led to the up-regulation of the genes involved in salvianolic acid biosynthesis, but inhibited the expression of the genes involved in tanshinone biosynthesis. Taken together, our results reveal SmGASA4 as a promising candidate gene to promote S. miltiorrhiza development. S. miltiorrhiza is considered the model Chinese medicinal plant. The demand for S. miltiorrhiza in both Asia and Western countries is large due to its documented therapeutic benefits. The roots and rhizomes of S. miltiorrhiza are used to treat neurasthenic insomnia, cardiovascular disease, cerebrovascular disease, hyperlipidemia, and acute ischemic stroke [S. miltiorrhiza can be divided into two groups, namely: (1) lipid-soluble tanshinones including tanshinone I, tanshinone IIA and tanshinone IIB, cryptotanshinone, dihydrotanshinone I; (2) hydrophilic phenolic acids, including rosmarinic acid, salvianolic acid B, lithospermic acid and dihydroxyphenyllactic acid, phenolic acid that displays antibacterial, anti-oxidative, and antiviral activity [S. miltiorrhiza is primarily concentrated on the surface of its roots. The biosynthetic production of these therapeutic compounds has been extensively studied in S. miltiorrhiza, but few reports have focused specifically on the root development [S. miltiorrhiza development is therefore important. Several external factors are known to influence S. miltiorrhiza root development, including the NH4+:NO3− ratio [Alternaria sp. A13, which markedly enhances S. miltiorrhiza root growth [S. miltiorrhiza growth in vivo are less well understood. In this regard, we previously identified mutant plants (MT) produced during the breeding experiments that possessed large taproots (2.85 times of the wild type (WT)) reduced lateral roots , the ratio of lateral root compare with taproot  and defective flowering, the MT with defect corolla and stamen, and can’t form normally. We reasoned that the identification of the genetic basis for this MT phenotype may reveal the genetic factors that contribute to root development which could be used to direct future S. miltiorrhiza breeding strategies [c stroke ,2. The aactivity ,4. The melopment ,6. Impro3− ratio  and the t growth . Howeverrategies ,10.S. miltiorrhiza that were designed to identify the biosynthetic processes that regulate the production of bioactive compounds in a range of tissues, or in response to specific induction stimuli [Several transcriptome analyses have been performed in  stimuli ,12. HoweArabidopsis) belongs to the family of cysteine-rich peptides (CRPs) [GAST1 (GA-stimulated transcript 1) was the first identified gene; gib1 is a GA receptor loss mutant and its expression was induced by the application of exogenous gibberellin [GASA gene family has also been identified in Petunia hybrid [Arabidopsis thaliana [Solanum tuberosum [Oryza sativa [GASA genes products play an important role in plant seed germination [GASA (Gibberellic Acid-Stimulated s (CRPs) . GASA iss (CRPs) , and is s (CRPs) . GAST1  components and 1–5 ABRE (Abscisic Acid Response Element), which participate in the GA and ABA pathways, respectively. This implies that the GASA expression is regulated by both GA and ABA [Populus root growth and development [GASA is a downstream target of GA [The residues ,29, whicresidues . In addiresidues , potentiresidues . The losfunction ,32. The et of GA , which iGASA1, GASA3, GASA4, GASA8, GASA10, and GASA14 promoters reveals their activity in root tissue and the vasculature, although through distinct spatial and temporal patterns. Roles in other tissues, including meristems, flowers, and seeds, have also been reported. AtGASA4 regulates the flower stem tissue [AtGASA4 in Arobidopsis reduces ROS (reactive oxygen species) accumulation and promotes GA activity during the H2O2 response [AtGASA14 in Arobidopsis also inhibits oxygen accumulation, and plants with gasa14 mutations show sensitivity to NaCl and ABA [AtGASA4 and AtGASA14 with cytoplasmic structures, implicating their involvement during plant growth [Analyses of the m tissue , promotem tissue ,18, posiresponse . The ovet growth .CRP (cysteine rich peptides) genes have been identified in 33 species, and 445 of these genes code for GASA/Snakin proteins [GASA family of genes are important to plants, their roles in S. miltiorrhiza remain poorly characterized. In this study, to reveal the mechanism of the MT phenotype and to identify new S. miltiorrhiza breeding genes, we employed RNA-seq to comparatively analyze the functional complexity of the S. miltiorrhiza transcriptome in WT vs. MT plants. We identified a GASA family gene termed SmGASA4, from the S. militiorrhiza transcriptome that encodes a 110-amino acid protein, the expression of which is inhibited in MT plants. Herein, we have introduced SmGASA4 into Arobidopsis and S. militiorrhiza under the control of a CaMV35S promoter, to verify its role during GA stimulation, drought, salt, and paclobutrazol (PBZ) stress, and as a secondary metabolism. We aimed to reveal the in vivo functions of SmGASA4 and its potential benefits to S. militiorrhiza breeding.Approximately 12,824 proteins . DespiteS. miltiorrhiza, we performed a transcriptome analysis of WT and MT plants. To enhance the data stability, biological repeats of each of the samples were performed. The samples were sequenced using an Illumina HiSeq™ 2500 with the production of 100 bp paired-end reads. After stringent data filtering and quality assessment, 27,255,174 clean paired-end reads were produced from the MT, as well as 30,598,611 reads from the WT plants  mapped reads from the MT plants, and 30,598,611 clean reads and 24,135,294 (78.87%) mapped reads from the WT plants .E-values less than 10−5 and HMMER  parameter E-values less than 10−10 to investigate the function. Once complete, the Unigene amino acid predictions were performed using HMMER software to compare the Unigene sequences with the Pfam libraries to achieve the Unigene annotated information. Among the 50,587 unigenes, 30,000 (59.3%) were annotated and 20,587 were not-documented. This may be due to technical limitations, including read length and sequencing depth, or the specificity of the S. miltiorrhiza genes. Amongst the annotated unigenes, 10,115 (20%), 20,532 (40.6%), 6337 (12.5%), 17,343 (34.3%), 21,674 (42.8%), 21,304 (42.1%), and 29,805 (58.9%) were annotated in the COG, GO, KEGG, KOG, Pfam, Swiss-Prot, and nr databases, respectively , Swiss-Prot, gene ontology (GO), Clusters of Orthologous Groups (COG), euKaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. We used BLAST software with parameter ectively .p-values. Using FDR values of <0.01, the fold-changes (FC) ≥ 2 were classed as differentially expressed genes. The comparison of WT and MT plants revealed 3714 up-regulated genes and 1371 down-regulated genes . We used the Benjiamini–Hochberg method to verify and revise the ed genes . Volcanoed genes a. MA  of proteins database is based on bacteria and algae. The eukaryotic system evolutionary relationships built using the COG database can classify the orthologous gene products. The top four categories for DEGs are (1) general function prediction only; (2) signal transduction mechanisms; (3) transcription; and (4) replication, recombination, and repair . These cWhen in vivo, the gene products perform their biological functions through a series of interactions. The DEGs identified were next assessed using the KEGG (Kyoto Encyclopedia of Genes and Genomes) libraries to determine their higher-level functions and utilities in the biological system. From the identified DEGs, the database included 38 terms for ‘plant hormone signal transduction’ and 35 terms for ‘plant-pathogen interactions’ , which iCL13560contigle (SmGASA4) expression was observed in the WT, compared to the MT plants (WT =1759 vs. MT = 173)  . Using NTo validate our expression profiling, 18 DEGs were randomly selected and their expression was assessed by qRT-PCR . Our results indicated identical patterns of expression for all of the 18 DEGs validating the transcriptome datasets .SmGASA4, plasmids for its exogenous expression in Arobidopsis and S. miltiorrhiza were produced. Under the control of the constitutive CaMV35S promoter, p35S::SmGASA4  , and the lateral root number and length were also larger in SmGASA4-S. miltiorrhiza   e. In then the WT f. The ovctively) a.SmGASA4 can promote Arobidopsis tolerance to drought. WT and SmGASA4-Arobidopsis plants were subjected to identical periods of drought stress, and the plant growth was assessed . When the plants were grown in a 400 mmol/L mannitol MS solid culture medium, both the WT and SmGASA4 growth and leaflet production were severely inhibited, but the root growths were prosperous , which tended to wither in these conditions.We next investigated whether the overexpression of assessed a. Duringhenotype b. The ovosperous . The oveSmGASA4 S. miltiorrhiza where compared . The WT plants had more abundant purple/yellow transitions in the bottom leaflets, shorter stems, and a reduced plant height compared with the SmGASA4 plants. Taken together, these data suggest that resistance to PBZ-induced plant death is conferred by SmGASA4 overexpression , class B (AP3 and PI), and class C (AG) floral organ identity genes in pattern discrete regions of the flower. LEAFY (LFY), AP1, and UFO genes promote the Arobidopsis floral meristem identity, and the increased branching phenotype of plants with reduced GASA4 expression are similar to those observed in LFY mutants [LFY and AP1 represent the key floral meristem identity genes, which specify lateral meristems to form flowers, but axillary inflorescent shoots [LFY is a key integrator of signals that promote flowering, including the gibberellin pathway [AP2 regulates the flower meristem and organ identity, and development in Arabidopsis. What is more, AP2 can also determine seed development, such as seed size, weight, and the accumulation of oil and protein in seeds, to a large extent [GASA4 and GASA6 is dysregulated in plants with altered LFY [GASA4 is involved in the regulation of hypocotyl elongation and flowering, in response to the integration of light signaling [GASA genes regulate flower meristem and flower organ development.The meristem identity genes activate class A , and repressing the expression of the key flowering genes, including flowering locus T (FT) and LFY [GASA4, GASA6, and GASA14 are known to promote plant development [GASA gene expression displays species, tissue, and period specificity, and GASA may function through its regulation of transcription factors, including the GA receptor DIG1 [GASA is also regulated by other phytohormones, including Auxin [GASA and LYF, AP1, AP2, AP3, PI, AG, UFO, FLC, FT, DIG1, GAI. and LFY . Howeverelopment ,48. The tor DIG1 , and GAIng Auxin  and ABA ng Auxin , amongstAtDREB1 gene families can significantly promote S. miltiorrhiza drought tolerance [Snakin/GASA proteins play an important role in plant resistance to abiotic stress, for example, the upregulation of PagGASA in poplar provides a high tolerance to drought stress [SmGASA4 promoted the Arobidopsis resistance to drought  pathway or through the modulation of SA  levels [GASA4 and Ca2+, IP3, DAG, PA, SOS, CBF/DREB1, DREB2, and ARF/AREB remains unclear [Drought is one of the greatest environmental constraints to agriculture worldwide ,52, The olerance ,36. Snakt stress . In this drought , an effeiorrhiza c. This rcterized . GASA co) levels ,55. Howe unclear ,57.Arobidopsis, the overexpression of AtGASA14 can promote salt stress resistance [OsGASR1 expression is induced by salt or ABA treatment, and the ectopic expression of OsGASR1 enhances salt resistance via the inhibition of ROS [SmGASA4 overexpression also promoted the salt resistance of Arobidopsis  accumulation, a function that is required for its GASA domain [FsGASA4, also enhances salt, oxidative stress, and heat stress tolerance during seed germination by means of increasing the SA biosynthesis. GA may thus play a crucial role during early plant responses to adverse environmental conditions by modulating the SA levels [SmGASA4 enhances salt resistance through a similar mechanism.In n of ROS . In thisbidopsis , and rediorrhiza d. The ovA domain . The oveA levels ,55. It iSnakin/GASA genes are involved in plant growth and stress tolerance by regulating DELLA expression [GASA1, GASA4, GASA6 and GASA9 through DELLA expression [GASA2, GASA3, and GASA4 upregulated in response to exogenous GA3 [SmGASA4 had a positive response to the GA3 treatment  protein [GEG; putative secretion; and the expression levels, which can also regulate cell wall division and extension [SmGASA4; OsGSR1 are downregulated by brassinosteroids (BR) [SmGASA4 and BR, GAI, and RGA should be further researched [The pression ,59. DELLpression  and comppression . GA regupression , with GAnous GA3 ,61. In treatment  in Arobif growth e. The GAe to GA3 ,61; GASA and ABA ; and thed by GA3 . In Arob protein . Gibberextension . GA, ABAids (BR) ,62; and searched .Arobidopsis seed germination, GASA14–1 plants showed an enhanced sensitivity to PBZ compared with the WT plants [SmGASA4 in Arobidopsis strongly promoted plant resistance to PBZ  and treated with RNase Free DNase I . RNA quality/purity was assessed using the Nanodrop 1000 spectrophotometer , and the RNA concentration and integrity assessed using a Qubit 2.0 Fluorimeter , and Agilent Bioanalyzer 2100 system . The mRNA was purified with Oligo (dT) magnetic beads. The fragmentation was randomly performed in an IIIlumina proprietary fragmentation Buffer. Using the mRNA as a template, random oligonucleotides synthesized the first-strand of cDNA, and the addition of dNTPs, RNase H, and DNA polymerase I synthesized the second-strand of cDNA, which was purified with AMPure XP beads. The purified double-stranded cDNA were enzymatically repaired, polyadenylated, and ligated to adapters. The fragment size selection was performed with AMPure XP beads and the cDNA libraries constructed through PCR amplification.TM2500  with PE100.Qubit 2.0 tests were used to assess the library concentration and the Agilent 2100 test was used to assess insert sizes. Q-PCR was used to assay the concentration of the libraries, to ensure quality. The cDNA library was sequenced using Illumina HiSeqThe raw data was filtered by removing the adapter sequence and low quality reads to produce high quality clean data. The sequence assemble of the clean data was assessed to produce Unigene libraries, most of the basic group quality score more than Q30, using the Trinity  softwarehttp://genome.ucsc.edu/cgi-bin/hgBlat) was used to compare the Unigene sequences with the non-redundant (nr) protein databases , Swiss-Prot (available online: http://www.uniprot.org/), gene ontology , Clusters of Orthologous Groups , euKaryotic Orthologous Groups , and Kyoto Encyclopedia of Genes and Genomes  libraries. Following prediction, the Unigene amino acid sequences were assessed using HMMER software (available online: http://hmmer.janelia.org/) for assignment into protein families , to obtain Unigene annotation information.BLAST software . The reverse transcription was performed using SuperScript III , following the manufacturer’s recommendations. A total of 18 DEG genes were randomly chosen to verify the RNAseq data  from Transcriptome cDNA, with primers containing the restriction sites NcoI/BstEII (SmGASA4 fragment was inserted into the binary plant vector pCAMBIA3301 containing the CaMV35S constitutive promoter (The full-length I/BstEII . Both ofpromoter . The Barpromoter . The conArabidopsis (Ecotype Columbia) transformation was carried out using the floral dip method [p method . We obta2 [−2 s−1). Each vector was transfered into Salvia miltiorrhiza by the leaf-disk method, mediated by Agrobacterium tumefaciens [A. tumefaciens, strain LBA4404, stored in 50% glycerol water, was transferred to a liquid yeast extract (YEB) medium for activation at 28 °C and cultured on a shaker (200 rpm) to mid-log phase (OD600 = 0.6). The excised leaves were pre-incubated on MS agar for 2 day at 25 ± 2 °C and 16-h-light/8-h-dark photoperiod.The leaves of the WT and MT plants were cultured on MS (Murashige and Skoog) + 1.0 mg/L 6-BA + 1.0 mg/L NAA  culture medium, containing 30 g/L sucrose and 6 g/L agar after surface sterilization with 0.1% HgCl2 . After tefaciens . The A. −2 s−1). The leaf disks were sub-cultured every week. The shoots were generated after 35 day and subsequently were excised and transferred to selective rooting medium. Two weeks later, the rooted plantlets were cut into sections and then cultured on ½ MS basal medium for propagation. A half-month-old seedling can be used for PCR screening and gene expression evaluation. The transformed plants were grown in the soil for further observations and stress treatment.The pre-cultured leaves were placed in the bacterial-containing liquid for 25 min, and the excess liquid was blotted on a sterile filter paper, and then placed the leaves on MS agar with 1.0 mg/L 6-BA + 0.01 mg/L NAA in the dark. The temperature was maintained at 25 ± 2 °C. After 2 day of co-cultivation, the leaves were placed on selective medium with 0.6 mg/L Basta and were cultured under specific conditions , following the manufacturer’s instructions, and were translated into cDNA. The primer sets for the p35S::SmGASA4 transgenic lines were verified using the 35S promoter sequence of pCAMBIA3301 , 22 ± 2 °C.To analyze the S. miltiorrhiza response to drought, NaCl, GA, and PBZ stress, as well as plantlets in solid sterile medium were transferred to sterile nutrient-containing soil, and then placed in a growth chamber for cultivation . Once the plantlets had adapted to their new environment, the stress treatments were initiated. Prior to drought stress, both the WT and transgene plantlets were saturated with water and left overnight to drain. The pants were sprayed with 250 mmol/L NaCl , 200 μmol/L GA3 (GA stress), or 300 μmol/L PBZ (PBZ stress). GA3 and PBZ were dissolved with ethyl alcohol prior to being dissolved in water.To analyze the SmGASA4, which displayed a reduced expression in the MT plants. The overexpression of SmGASA4 was subsequently shown to promote root development and promote flower and capsule development in Arobidopsis. We further demonstrate that SmGASA4 is a GASA family member that is positively regulated by GA. SmGASA4 expression was also shown to improve plant resistance to drought, salt stress, and PBZ. Finally, we demonstrate that the overexpression of SmGASA4 in S. miltiorrhiza can upregulate the key genes of the salvianolic acid biosynthesis, but down regulates the genes involved in tanshinone biosynthesis.Our laboratory produced a mutant plant that possessed a single huge taproot, reduced lateral roots, and defective flowering. By performing a transcriptome analysis, we found that phytohormone signaling may play an important role during WT plant growth. We identified a significant DEG,


This work describes, to our knowledge, the first NMR metabolomics analysis of mice kidney, liver, and breast tissue in response to cisplatin exposure, in search of early metabolic signatures of cisplatin biotoxicity. Balb/c mice were exposed to a single 3.5 mg/kg dose of cisplatin and then euthanized; organs  were collected at 1, 12, and 48 h. Polar tissue extracts were analyzed by NMR spectroscopy, and the resulting spectra were studied by multivariate and univariate analyses. The results enabled the identification of the most significant deviant metabolite levels at each time point, and for each tissue type, and showed that the largest metabolic impact occurs for kidney, as early as 1 h post-injection. Kidney tissue showed a marked depletion in several amino acids, comprised in an overall 13-metabolites signature. The highest number of changes in all tissues was noted at 12 h, although many of those recovered to control levels at 48 h, with the exception of some persistently deviant tissue-specific metabolites, thus enabling the identification of relatively longer-term effects of cDDP. This work reports, for the first time, early (1–48 h) concomitant effects of cDDP in kidney, liver, and breast tissue metabolism, thus contributing to the understanding of multi-organ cDDP biotoxicity. This Regarding the use of metabolomics to improve the understanding of cDDP biotoxicity mechanisms, some studies have addressed healthy liver , lung 1, and kid1H NMR-based metabolomics strategy was carried out, for the first time to our knowledge, to assess the impact of cisplatin on the metabolism of the kidney, adding the novel study of the concomitant metabolic changes in the liver and breast tissues of the same healthy mice. The latter type of tissue should be particularly useful as a control matrix for studies of breast cancer animal models treated with cisplatin or other drugs. As a technique which is complementary to MS-based methods, NMR metabolomics should confirm previous observations in kidney tissue , while unveiling novel metabolic changes, not only in kidney, but also in liver and breast tissue. Focusing on a very short timescale of exposure to cDDP (1 to 48 h), this study provides an early dynamic metabolic picture of the interplay of different organs in responding to the drug, thus unveiling potential early multi-organ metabolic markers of cDDP biotoxicity.In this work, a 1H NMR spectra of polar extracts obtained from the kidney, liver, and breast tissue of healthy mice (controls), including semi-quantitative indication of predominant metabolites (compounds noted in red). 1H NMR spectra of kidney [N-oxide (TMAO), uridine diphosphate-glucuronate (UDP-GlcA)), liver , hypoxanthine, hippurate, IMP, threonine, 3-HIBA, TMAO, and UDP-GlcA), and breast tissue, for which, to our knowledge, no previous 1H NMR metabolomics study has been reported. By visual inspection of the spectra in m-inositol, and inosine, (2) liver shows high levels of acetone, glucose, glycogen, and glutathione , and (3) breast tissue shows high levels of creatine, phosphocholine, and glycerol moieties arising from glycerolipids. The following section describes the changes in the complex metabolic profiles of each tissue type upon time-course exposure to cDDP.f kidney ,45,46 anf kidney ,48,49, a2 = 0.48, compared to Q2 = 0.38 and 0.30 for liver and breast tissue, respectively). Particularly for kidney samples, it is interesting to note, through examination of PLS-DA plots, that both control and exposed kidney samples seemed to follow a slight time course tendency towards positive LV2, with relatively larger sample dispersion for the drug-exposed group.Unsupervised analysis with PCA of the spectra of both controls and cDDP-exposed groups showed group separation only in the case of kidney , left, r2 = 0.73), together with sample groups for 1 h , both showing better separation than for 48 h , which seems to indicate some degree of recovery from the shorter-term impact of cDDP. The underlying varying metabolites were identified through inspection of the LV1 loading plots corresponding to each time , phosphocholine (PC) , taurine , trimethylamine (TMA) , and UDP-GlcA   see .m-inositol (see +), betaine, and UDP-GlcA. The column corresponding to 48 h shows that many metabolites approach levels characteristic of controls, as illustrated by white or light colors without statistical relevance (m-inositol.The shorter-term (1 h) effects of cDDP on kidney comprise a general decrease in amino acids, with the exception of taurine (which increases significantly), in tandem with decreased free choline, and increased levels of ADP/AMP/UMP, 3-HIBA, and itol see a. Mediumelevance . More pe2 = 0.70)  b, and th = 0.70) b, right +, and four unassigned resonances, in tandem with an acetate decrease.The current results show that the short-term effects of cisplatin in liver are limited to decreases in lactate and threonine (as previously reported for human liver cell line L02 exposed to cDDP ), to whiBreast tissue is shown to exhibit the least affected metabolic profile following treatment with cisplatin c, with nRegarding the effect of cDDP on mouse organ metabolism, this work is, to the best of our knowledge, the first NMR-based metabolomics study. In the case of kidney metabolism, as a complementary strategy to MS-based metabolomics , this NMR work complements previous MS studies of mice kidney extracts carried out to characterize cDDP effects ,42,43. Fm-inositol, adenine, and uridine nucleotides, namely ADP/AMP/UMP, the latter in apparent contradiction to the recent high-dose cDDP report [m-inositol, NAM (vitamin B3), and TMA remain at 48 h as relatively late effects of cDDP. The latter two compounds may be indicative of an abnormal gut microflora metabolic response to cDDP, consistent with previous reports of dysbiotic intestinal microflora in relation to kidney disease [In terms of the relative dynamics of the metabolic effects, it was observed that kidney suffers a large impact of the drug at only 1 h, evidencing the strongest short-term effect within the different tissues considered. At 1 h, the general amino acids decrease noted in kidney tissue is consistent with previous reports that employed cDDP doses of similar orders of magnitude , altP report , consistP report . Deviant disease .+ noted at 48 h post-injection. The profile in unassigned resonances shows a general reversal from 12 to 48 h, which in itself is interesting as part of the characteristic signatures, thus supporting further endeavors to identify these resonances. In the case of liver, this is, to the best of our knowledge, the first NMR metabolomics study of liver tissue exposed to cDDP; the only comparison possible with previous results refers to the human liver L02 cell line [The early amino acid depletion in kidney is accompanied by a qualitative (non-significant) tendency for amino acids decrease also in liver, although statistically relevant effects are few, and are related to decreased lactate levels and a few other unassigned resonances. The major impact of cDDP on liver takes place at 12 h and 48 h, but in a distinct way: at 12 h, ketone bodies (acetone and 3-HBA) seem to be used up for enhanced energy production; this may justify the enhanced levels of AMP/ATP/IMP/NADell line . In cellFinally, in tandem with the early effects on kidney and liver discussed above, the sole early change in breast tissue (increased ketone body 3-HBA) may reflect a deviant shift to enhanced fatty acid oxidation which quickly leveled off subsequently. The strong nitrogenated bases decrease at 12 h, and the usage of glutamine at 48 h requires further investigation, although we propose at this point that nucleotide metabolism and glutamine usage, possibly to sustain energy production, may contribute to the metabolic breast tissue signature of cDDP response.® solution  was from Le Vet . All reagents were of analytical grade.Cisplatin (cis-dichlorodiammine platinum (II), 99.9%) was purchased from Sigma-Aldrich . EuthasolHandling and care of animals were consistent with Portuguese (Decreto-Lei n.°113/2013) and European legislation (Directive 2010/63/EU) on the protection of animals used for scientific purposes, and were in agreement with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health (NIH). The study protocol was approved by the Ethics Committee for Animal Experimentation of the Faculty of Pharmacy of the University of Porto, Porto, Portugal (Permit Number: 25-10-2015), and by the Ethics Committee and the Organ Responsible for the Welfare of Animals of ICBAS-UP, Porto, Portugal (Permit number 134/2015). The ARRIVE Guidelines were followed for reporting in vivo experiments .ad libitum access to water and standard pellet food under controlled 12 h light/dark cycles (lights on at 7.00 AM), temperature (22 ± 2 °C), and humidity (50 ± 10%).Six-week old, Specific-Pathogen-Free (SPF), female BALB/cByJ mice  were purchased from Charles River Laboratories  and acclimatized for 1 week at ICBAS-UP Rodent Animal House Facility . Animals were randomly distributed in groups of five per cage  with enrichment material , and were housed in a SPF environment with 2PO4 1.5 mM, Na2HPO4 4.3 mM, KCl 2.7 mM, NaCl 150 mM, pH 7.4 and PBS alone were administered in a single dose, via intraperitoneal injection (volume of injection = 200 µL) in the cDDP and vehicle groups, respectively. After drug/vehicle administration, five animals from each group were euthanized, at each time-point , with pentobarbital intraperitoneal injection (120 mg/kg) followed by cardiac puncture. The left kidney, median liver lobe, and thoracic mammary glands were excised and snap frozen in liquid nitrogen and stored at −80 °C for NMR analysis. All injected solutions were sterile filtered. No biochemical measurements were performed to detect kidney damage, as the volume of blood obtained was too small . In any case, all animals showed no loss of weight , no evidence of decreased food or drink intake, and no behavioral changes whatsoever, which indicated the absence of significant kidney damage. This was also supported by identical observations in a small pilot study (n = 2) of our own with animals injected with 3 cycles of cDDP (3.5 mg/kg per cycle), and by a previous report that blood urea nitrogen (BUN) in mice increased after 24 h for a 30 mg/kg b.w. cDDP dose [After 1 week of acclimatization, animals were randomly allocated into two groups : cDDP and vehicle (PBS). At the beginning and end of the experiments, the animals weighed 19.9 ± 1.5 g and 20.0 ± 1.6 g, respectively. CDDP 3.5 mg/kg solution in PBS: Hca. 50, 60, and 35 mg for kidney , liver (median liver lobe), and breast tissue (predominantly consisting of mammary gland), respectively) and ground by mechanical maceration with cooled liquid N2 using a pestle and mortar [2O containing 0.25% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid ), homogenized, and 600 µL of the resulting solution was then transferred to NMR tubes.Tissues were weighted  proton NMR spectra of the aqueous extracts were recorded at 298 K, using the “noesypr1d” pulse sequences (Bruker library), with 2.34 s acquisition time, 2 s relaxation delay, 512 scans, 7002.801 Hz spectral width, and 32 k data points. Each free-induction decay was zero-filled to 64 k points and multiplied by a 0.3 Hz eponential function prior to Fourier transformation. Spectra were manually phased, baseline-corrected, and chemical-shift referenced to TSP. Bidimensional (2D) NMR homonuclear (TOCSY) and heteronuclear (HSQC) spectra were recorded for selected samples to aid spectral assignment. Peak assignment was based on comparison with data available on Bruker BBIOREFCODE spectral database and the human metabolome database (HMDB) [All NMR spectra were acquired on a Bruker AVANCE III spectrometer operating at 500.13 MHz for e (HMDB) , as well2) values ≥ 0.05. The relevant resonances identified in PLS-DA loadings plots were integrated , normalized, and variations assessed by univariate analysis  . Significantly changed metabolites (p < 0.05) were identified, and the corresponding effect size values calculated [p-values for multiple comparisons.The 1D NMR spectra were converted into data matrices  prior multivariate analysis, and exclusion of the water (δ 4.5–5.2) and methanol (singlet at δ 3.36) regions was applied. Spectra were aligned by recursive, segment-wise peak alignment (RSPA)  and normalized to spectral total area, to account for sample concentration differences. Multivariate analysis of the 1D spectra was carried out using principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) after unit variance (UV) scaling of the spectra . PLS-DA loadings were back-transformed, multiplying each variable by its standard deviation, and colored according to variable importance to the projection (VIP) . PLS-DA models were considered statistically robust for predictive power . Liver and breast tissue also show more marked metabolic changes at 12 h, with liver standing out for its persistent increase in the levels of several nucleotides as a longer-term cisplatin effect. The present study, however, presents some limitations, namely regarding some still unassigned resonances included in all tissue signatures , and the lack of biochemical measurements of biotoxicity in both kidney and liver tissues . In spite of these, this work successfully reports the very early (1–12 h) cisplatin effects in kidney, liver, and breast tissue metabolism, thus contributing to our understanding of multi-organ cDDP biotoxicity.This work describes, to the authors´ knowledge, the first NMR metabolomics analysis of mice kidney, liver, and breast tissue in response to cisplatin exposure (single 3.5 mg/kg injection), to search for early metabolic signatures of cDDP biotoxicity. The results obtained show that the largest metabolic impact occurs for kidney, as expected, as early as at 1 h post-injection. Kidney shows a marked depletion in amino acids, comprising a 14-metabolite signature at 1 h. The highest number of variations is noted at 12 h after drug administration, although many of these recover to control levels at 48 h, with the exception of persistent changes in 10 specific metabolites (namely, alanine, BCCAs, tyrosine, hypoxanthine, fumarate


There is also growing evidence indicating that the LPA–LPAR axis also plays an important role in inducing pathological alterations of cell structure and function in the kidneys. In this review, we will discuss key mediators or signaling pathways activated by LPA and summarize recent research findings associated with DN.Lysophosphatidic acid (LPA) is a bioactive phospholipid present in most tissues and body fluids. LPA acts through specific LPA receptors (LPAR1 to LPAR6) coupled with G protein. LPA binds to receptors and activates multiple cellular signaling pathways, subsequently exerting various biological functions, such as cell proliferation, migration, and apoptosis. LPA also induces cell damage through complex overlapping pathways, including the generation of reactive oxygen species, inflammatory cytokines, and fibrosis. Several reports indicate that the LPA–LPAR axis plays an important role in various diseases, including kidney disease, lung fibrosis, and cancer. Diabetic nephropathy (DN) is one of the most common diabetic complications and the main risk factor for chronic kidney diseases, which  Lysophosphatidic acid (LPA) is a small, naturally occurring glycerophospholipid, which is composed of a glycerol backbone with an ester-linked acyl chain and a phosphate group. It is also produced by the action of various lysophospholipases, including autotaxin (ATX), and phospholipases A1 or A2 (PLA1 and PLA2). LPA acts through specific LPA receptors (LPAR1 to LPAR6) coupled with G protein and is associated with a wide range of cell responses, such as proliferation and migration ,6, and pIn this review article, we review the key mediators and signaling pathways activated by LPA and summarize newly reported signaling pathways modulated by different LPA receptor antagonists in different DN models.sn1 (saturated) or sn2 (unsaturated) positions of PA [LPA is the smallest bioactive lysophospholipid (MW 430–480 Da) derived from membrane phospholipids  and actsns of PA ,18. Membns of PA ,20.LPA is converted by several classes of enzymes, including lipid phosphate phosphatases (LPPs), LPA acyltransferase, and phospholipases ,22,23, aEdg) family, and LPAR4 to LPAR6 belonging to the P2Y purinergic gene cluster [2+ mobilization, PLC, adenylyl-cyclase inhibition, and MAPK activation [2+ levels [2+ responses by LPA stimulation. LPA can also bind to and activate non-GPCR targets, the receptor for advanced glycation end products (RAGE) [KB, serum response factor (SRF), PI3K, and Akt [2+ levels following LPA stimulation [LPA induces various cellular effects by binding to specific G protein-coupled LPA receptors (LPARs) and activates downstream intracellular signaling pathways, resulting in various physiological and pathophysiological responses . Till no cluster . These r cluster ,28. LPARtivation ,30. LPARtivation . LPAR5 i+ levels . LPA6/P2+ levels ,33. In a+ levels  and P2Y1+ levels , were als (RAGE) , and thes (RAGE) . RAGE pa and Akt . TRPV1 imulation . Anothermulation . Among tmulation . AlthougKidney disease is a heterogeneous group of disorders affecting kidney structure and function, which can be further classified into two distinct syndromes, acute and chronic kidney disease (CKD) . Acute kGlomerulosclerosis is hardening of glomeruli in the kidney and is frequently referred to as focal segmental glomerulosclerosis or nodular glomerulosclerosis . The iniAlthough the initial glomerular damage and glomerulosclerosis are the major factors for the development of CKDs, the fibrosis in the tubulo-interstitial compartment is also a major contributor ,49. InitIn the diabetic milieu, such as hyperglycemia and dyslipidemia, metabolic alteration increases the production of ROS mainly through the Polyol and/or Nox pathway ,57. In aThe Smad signaling pathway has also been reported to be associated with the renal hypertrophy and accumulation of ECM molecules through TGF-β signaling in DN ,60,61. ARecently, new molecules or potential signaling pathways have been added to the established pathways, which has made the pathogenesis of DN more complex. However, new observations simultaneously increase the possibility of identifying new targets for the treatment of DN. Interleukin (IL)-33-mediated suppression of the tumorigenicity 2 receptor (ST2) axis is one such pathway, which is very attractive due to its association with inflammatory response. IL-33 was first identified as a member of the IL-1 family in 2005 . IL-33 aSodium–glucose cotransporter (SGLT)-2 may be a potential contributor to CKD. Under normal physiological conditions, urine does not contain glucose, owing to its effective reabsorption by two transporters, SGLT-1 and SGLT-2 . The SGLIn the diabetic milieu, increased oxidative stress, inflammation, and mitochondrial dysfunction modulate the autophagy activation and inhibition as well as lead to cellular recycling dysfunction [Current evidence also suggests that autophagy is critical in kidney physiology and homeostasis. function . Reductifunction ,75. Highfunction . Similarfunction ,78. Autofunction . Severalfunction ,81,82.db/db mice, a robust model of DN [CKD has been becoming a major public health problem globally. It involves a progressive loss of kidney function over a period of months or years, which frequently leads to end-stage renal failure . The maiel of DN . Furtherel of DN . More reel of DN . Consistel of DN . On the el of DN ,89,90. Lel of DN .db/db mice (leptin receptor-deficient mouse with human type 2 diabetic phenotype) and streptozotocin (STZ)-induced diabetic mice (pancreatic beta cell-destroyed mouse with human type 1 diabetic phenotype) [db/db mice [db/db mice. However, treatment of ki16425 reduces proteinuria, glomerular tuft area and volume, and mesangial matrix expansion by regulating the LPA–GSK3β–SREBP1 axis [Recently, we and another group reported that the LPAR1 and/or LPAR3 were upregulated in different DN mouse models ,92,93. Tenotype) ,92,93. T/db mice . LPA–LPA/db mice . SubsequBP1 axis . In lineBP1 axis . AdditioBP1 axis . All of db/db mouse [Similarly, Zhang et al. showed improvements in renal function, using a different dual LPAR1/3 antagonist, BMS002, in the endothelial nitric oxide synthase-deficient (eNOS (–/–)) db mouse . The expdb mouse . Blockaddb mouse , which pdb mouse . HoweverFurthermore, our studies showed that increased LPA levels in STZ-induced diabetic mice increases Toll-like receptor (TLR) 4 expression and directly activates the NF-κB, the master transcription factor responsible for inflammatory cytokine expression. Simultaneously, activated TLR4 also produces ROS through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system . All theKip1. The expression of Krüppel-like factor 5 (KLF5) was upregulated by activating MAPK and elevating the expression of early growth response 1 (Egr1) in the kidney cortex of db/db mice and LPA-treated SV40 MES13 cells. Moreover, LPA significantly increased the activity of Rac1 GTPase in SV40 MES13 cells, and the dominant–negative form of Rac1 blunted the phosphorylation of p38, the upregulation of Egr1, and the LPA-mediated induction of KLF5, indicating that the downstream pathway of Rac1 was involved in LPA-induced mesangial cell proliferation [Mesangial cell proliferation and accumulation in the pathogenesis of DN is a major risk factor contributing to glomerulosclerosis. LPA treatment increased the proliferation of mouse mesangial cells (SV40 MES13), concomitant with the increased expression levels of cyclin D1 and CDK4 and decreased expression of p27feration . Taken tferation . Severalferation ,99. ThusSome studies have also suggested that LPA may bind to the receptor for RAGE, a member of the immunoglobulin superfamily ,100. UndDN is the main risk factor for chronic kidney diseases, which mostly progress to the development of ESRD in the end. The best treatment regimen for DN is kidney replacement; however, it is highly restricted, due to various reasons, such as rare donors and rejection of the transplanted organ. Several medications are currently available for DN, but they are not sufficient for the recovery from kidney injury and restoration of kidney function in DN patients. Alternative drugs are thus required. Accumulated evidence indicates that the LPA–LPAR axis plays an important role in inducing pathological alterations of cell structure and function in the kidneys. Current studies showed that LPAR antagonism using pharmacological inhibitors significantly decreases the abnormality of kidney structure, such as GBM thickness, and increases of renal function, such as reducing proteinuria in different types of diabetic mouse models by regulating several signaling pathways, as shown in KB/MAPK or TLR4-NF-KB/MAPK axis for inflammatory response; 4) PI3K-AKT-GSK3α axis for apoptosis; 5) LPA-RAGE axis for glomerular injury; and 6) Wnt/β-catenin axis for fibrosis and apoptosis. For further details, please see above. LPAR: LPA receptor, RAGE: AGE receptor, Rac1: Ras-related C3 botulinum toxin substrate 1, NADPH: Nicotinamide adenine dinucleotide phosphate oxidase, TLR4: Toll-like receptor 4, NF-KB: Nuclear factor-KB, KLF5: Krüppel-like factor 5.The currently available data suggest that LPA signaling may regulate fibrosis, proliferation, and the inflammatory response in mesangial cells and podocytes or induce apoptosis via the following signaling pathways: 1) PI3K-AKT-GSK3β-TGF-β axis for fibrosis; 2) Rac1GTPase-MAPK-KLF5-CDK4/Cyclin D1 axis for proliferation; 3) TLR4-NADPH oxidase-ROS–NF-However, there are still many unanswered questions. Further studies should be performed in the following directions: Since the kidney consists of heterogeneous cells  and the LPAR expression level may be different in different cell types, further studies should first determine the LPAR expression level under different physiological and pathophysiological conditions. In addition, the studies should investigate the effects of LPAR antagonism on each type of kidney cell, using not only pharmacological inhibitors, but also genetically engineered in vivo models. Moreover, there are many different forms of LPA, which exhibit different affinities for each LPAR type. Each LPAR also exhibits various interaction affinities with one or more G protein subunits, thereby promoting different biological effects. These aspects should be further elucidated. Furthermore, the LPA–LPAR-mediated signaling pathway is also modulated by a cross-talk with other signaling pathways, such as epidermal growth factor (EGF) or vasoactive (angiotensin II) receptor-mediated signals , which w


Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn ( Macrobrachium nipponense) is a useful test organism for laboratory-based hypoxia research because it is small and reproduces rapidly. Recent studies in our laboratory showed that hypoxia causes energy metabolism disorders in M. nipponense brain tissue [Hypoxia is a natural phenomenon in aquaculture ponds, especially in summer, that negatively impacts aquatic animals by limiting behavior, growth, reproduction and even survival . Orientan tissue . Althougn tissue , the moln tissue ,5,6, it ATGs) present in yeast and higher eukaryotes [ATGs have been cloned and characterized from fish, including Beclin1 in Gobiocypris rarus [Paralichthys olivaceus [Cyprinus carpio) [ATG5 in Danio rerio [ATG4 in Pelteobagrus fulvidraco [Macrobrachium rosenbergii transcriptome data and examined the presence of key ATG proteins in tissues using western blotting [ATG mRNA expression profiles have not been reported.Transportation of cytoplasmic cargo to lysosomes is dependent on autophagy since this process contributes to homeostasis of organelles and proteins . Autophakaryotes , but onlis rarus , Paraliclivaceus  and comm carpio) , ATG5 inio rerio  and ATG4lvidraco . A recenblotting . HoweverBeclin1/ATG6 complex and ATG7-ATG3-ATG5 ubiquitin-like conjugation, MAP1LC3 conjugation and ATG9 vesicle recycling systems  reportedly involved in autophagosome expansion and membrane elongation, and explored their expression in different tissues and developmental stages in juvenile prawns, under normal and hypoxia conditions. The findings expand our understanding of autophagy and hypoxia stress at the molecular level, and illuminate the adaptive protective mechanism of autophagic responses to hypoxia in crustaceans.In the present study, we investigated autophagy in the brain of ATG3, ATG4B, ATG5, and ATG9A fragments yielded cDNA sequences of length 1652 bp, 1512 bp, 1731 bp and 3072 bp respectively , including open reading frames (ORFs) encoding proteins of 319, 264, 268 and 828 amino acids respectively . None of the genes were predicted to include a signal peptide according to SignalP 4.0 Server . The deduced polypeptide encoded by the M. nipponense ATG3 cDNA encodes a typical conserved active-site domain and a flexible region  of the e region . M. nipp residue . M. nippge sites . M. nipp helices . Phylogeionships , which wM. nipponense ATG3, ATG4B, ATG5, and ATG9A in different tissues and developmental stage was investigated by qRT-PCR, and mRNAs of all four genes were widely expressed in hepatopancreas, gill, muscle, brain, heart, and intestine, with highest expression in brain and hepatopancreas (p < 0.05) than the post-larval stage , whereas ATG3 and ATG5 mRNA levels in brain were only slightly elevated (p < 0.05) after 12 h and 24 h of hypoxia. However, no significant differences in the ATG9A mRNA expression levels were observed  in brain after 6 h, 12 h and 24 h of hypoxia than levels in normoxia groups , pyruvate kinase (PK), and lactate dehydrogenase (LDH) enzymes were significantly higher  in brain tissue of control prawns not injected with dsRNA. By contrast, ATG8 expression decreased by 68% or 72% after 24 h under normoxia or hypoxia conditions in the brains of dsRNA-injected prawns when ATG4B is absent (Oncorhynchus mykiss) [ATG4B nor dsEGFP affected cell viability in M. nipponense juveniles prior to hypoxia exposure. However, following ATG4B knockdown, there was a significant (p < 0.05) reduction in cell viability in ATG4B-depleted prawns compared with dsEGFP-injected prawns after 24 h of hypoxia  . Injecti hypoxia D. Our reATG4B (rMnATG4B) and ATG8 (rMnATG8) from M. nipponense were successfully expressed and purified in our laboratory. GST pull-down assay was carried out to test the binding ability of recombinant MnATG8His and recombinant MnATG4B-GST, which were subjected to 12% SDS-PAGE. The experimental results demonstrated that rMnATG8 could interact with rMnATG4B in M. nipponense, but not with the GST-tag protein  [According to the literature, most studies in mammalian cell lines indicates that that hypoxia positively regulates autophagy ,20,21,22activity ,24. Cys-y [ATG4B , and the Trp-142 . M. nippurvature . M. nipph domain . Autophain [ATG9 , and M. te (PAS) .ATG mRNAs were found to be expressed in multiple M. nipponense tissues, with higher expression levels in brain and hepatopancreas, indicating the potential for tissue-specific regulatory mechanisms, as reported previously in mammals [ATGs in all prawn developmental stages, with higher levels in earlier larval stages, consistent with previous studies in insects and prawns [In the present study, all four  mammals ,32. The d prawns .M. nipponense [ATG3, ATG4B, and ATG5, all of which may play significant roles in autophagy-mediated regulation of cellular adaptive responses to hypoxia in M. nipponense brain tissue. Furthermore, we observed that ATG4B is localized in diverse types of neuronal cells (including secretory and astrocyte cells) in the brain; this means that it might play an important role in the cerebral ganglion of prawns in response to hypoxia. Thus, autophagy may be important for the prevention of neural degenerative diseases [Autophagy is critical for homeostasis in brain cells, and is tightly regulated under normal conditions . Recent pponense ,3. Therepponense ,36,37. Cdiseases ,38.ATG4B. In a previous study on M. nipponense, we demonstrated that the effects of RNAi can last 5 days [ATG4B caused a significant decrease in ATG8 transcription in the prawn brain, and decreased brain cell viability, suggesting that autophagosome biogenesis is dependent on processing of ubiquitin-like ATG8 proteins by cysteine protease ATG4. There is some evidence that yeast ATG4 is recruited to autophagosomal membranes by direct binding to ATG8 via two evolutionarily conserved ATG8 recognition sites [ATG4B and ATG8 were examined. The results of the GST pull-down assay showed that rATG8 could specifically interact with rATG4B (GST tag) in prawns. Thus, we speculated that ATG4 functions as a constitutive ATG8-binding module.In this present work, RNA interference (RNAi) was used to probe the function of t 5 days . Given tt 5 days , and emet 5 days , we wereon sites . Thus, tExperimental protocols for hypoxia challenge were performed as previously reported . Prawns ATG3, ATG4B, ATG5, and ATG9A cDNAs were performed as described previously [Procedures for cloning eviously . Primersβ-actin housekeeping gene as an internal transcriptional reference [ATG3, ATG4B, ATG5, and ATG9A was calculated using the 2−ΔΔCt method [Total RNA was extracted from different tissues, from different growth stages, and from prawns cultured under control and hypoxia conditions using RNAiso Plus Reagent . Analysis of mRNA expression levels was performed by quantitative real-time PCR (qRT-PCR) as described with the eference . Primerst method .ATG4B was obtained and used for probing according to a previously described method [The eyes and brains were dissected from the prawn, as described above, and fixed in 4% paraformaldehyde in phosphate buffer saline  at 4 °C overnight. Chromogenic in situ hybridization (CISH) was performed on 4-μm thick formalin-fixed paraffin-embedded sections using the Zytofast PLUS CISH implementation kit , as reported in a previous study . The slid method .ATG4B dsRNA via the carapace pericardial cavity membrane [n = 60) were injected with an equal amount of dsEGFP dissolved in the injection buffer.In vitro synthesis of dsRNA was performed as previously described , and 120membrane . ControlCells were obtained from brain tissue homogenates as described previously , and celATG4B (MnATG4B) and ATG8 (MnATG8) from M. nipponense were expressed in E. coli as His-tagged fusion proteins using the pET-28a  expression system, as described in our previous study. MnATG4B and MnATG8 were purified using the GST-Bind resin. The pull-down assay was performed as previously described [GST protein was used as a negative control in this assay.Recombinant escribed . After tp < 0.05 was considered statistically significant.Results are presented as the mean ± standard error. Statistical analysis was performed using Statistical Package for the Social Sciences (version 19.0). Different tissues and developmental stages were compared using one-way analysis of variance and Duncan’s multiple range tests. Treatment and control groups were independently compared with Student’s t-tests, and ATG3, ATG4B, ATG5, and ATG9A from M. nipponense, and determined their expression profiles in different tissues and developmental stages. Hypoxia up-regulated the expression of the autophagy-related genes ATG3, ATG4B, and ATG5 at the transcriptional level in prawn brain tissue. In vivo silencing of the AG4B gene to prevent autophagy resulted in a decrease in brain cell viability, and recombinant MnATG4B exhibited binding activity with the rMnATG8 protein. These findings reveal that the fundamental mechanisms of autophagy involve putative adaptive catabolic processes that are activated in response to hypoxia.In summary, we cloned and characterized full-length cDNAs of


The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases  Developing human peripheral sensory neurons (PSNs) are of great interest, not only to advance pain research aiming at finding new analgesics, but also to the cosmetic industry to develop alternative  methods . In the  methods . Capsaic methods .Transient receptor potential channels comprise a diverse family of ligand-gated, mostly non-selective, cation channels that are robustly expressed in sensory systems throughout species . Of thesSome attempts were made to generate somatosensory neurons from hiPSCs, but so far, these cells have been proved to be difficult to generate robustly. For instance, Chambers and collaborators developed a differentiation protocol based on a small molecule screen and successfully obtained neurons that express the main expected markers . NeverthKeratinocytes and sensory neurons have an extensive interplay during development and within mature skin. For instance, keratinocytes release neurotrophic factors that induce branching of free nerve endings and neurite outgrowth toward the skin surface . They alIn the glabrous skin, the sensory information is processed by four types of mechanoceptors: Merkel cells, Pacinian corpuscles, Meissner and Ruffini terminations. In the hairy skin, tactile transmission is associated with the lanceolate Aβ-LTMR terminations. Hair follicles are innervated by the longitudinal lanceolate terminations C-LTMR, Aδ-LTMR, and Aβ-LTMR. The intercommunication between nerve endings and epidermal keratinocytes occurs through neuropeptides involved in nociceptive and pruriceptive sensitization .Substance P (SP) is a neuropeptide, member of the tachykinin family, synthesized by sensory neurons that emit their extensions from the dorsal root ganglion (DRG) to the more superficial layers of the skin, mediating the communication between peripheral neurons and epidermal keratinocytes . Most ofLiterature analysis reveals the efforts made by several groups to develop more efficient and cost-effective protocols for obtaining functional peripheral neurons from hiPSCs . HoweverIn the present work, we developed a new protocol for the direct differentiation of hiPSCs to PSNs in the presence of conditioned medium (CM) obtained from HEKn. These neurons showed some expected functional responses, such as neuropeptide release. The analysis of differential gene expression confirms the transcriptional profile of putative human somatosensory peripheral neurons.Supplementary Figure 1).Human induced pluripotent stem cells  were cultured on matrigel-coated plates with mTeSR . The cells were checked for pluripotency markers expression such as Nanog, Sox2, Tra-1-60, and Tra-1-81 (data not shown), and were negative for the neuronal markers Islet1, BRN3A, peripherin, and TRPV1 . When the same confluence was achieved again, which could take between 1 and 2 days, the differentiation was initiated by switching to 3N medium  containing 500 nM LDN-193189  and 10 μM SB431542 . In the following day (d2), the same two factors were added again, together with 3 μM CHIR-99021  . On day 3 (d3), the medium was changed to have LDN removed and on day 4 (d4) SB was not added, maintaining just CHIR until d10, with medium changes at odd days. Treatment with these Smad pathway inhibitors leads to differentiation of hiPSCs cells toward neural crest progenitor cells (NCPCs) . On day 11 (d11) of differentiation, NCPCs were plated onto Polyornithine (PLO)/Laminin-coated plates and maintained in 3N medium for neuronal differentiation with addition of 10 ng/ml FGF-2  and 10 ng/ml EGF  for approximately 2 days for expansion. Then, the cells were split again and plated to be differentiated into neurons, when 80% confluency was reached (between 1 and 2 day). From about d13 to d33 of differentiation, the cells were cultured in 3N medium supplemented with 10 ng/ml BDNF , 200 μM ascorbic acid (AA) , 10 ng/ml GDNF , 10 ng/ml NGF , 10 ng/ml NT-3  and 0.5 mM cAMP , resulting in the formation of immature PSNs. After these 35 days, cells could be replated after enzymatic passage with Accutase  and incubated with the same neuronal medium supplemented with 10 μM iRock (Millipore). Subsequently, after 1–2 days, neurons were kept for additional 2, 5, or 10 days in CM from neonatal human epidermal keratinocytes (HEKn), obtained as described in the following session.When approximately 60–70% confluence was reached, cells were split using a 0.5 mM EDTA solution in PBS     and HEKn  were cultured in Epilife medium . Cells were split when 70% confluence was reached. After 48 h conditioning, medium was collected, centrifuged to remove debris and dead cells and was added fresh to the neurons at 75% with 25% of neuronal media.Supplementary Datasheet 1 of the Supplementary Material. Negative controls for immunocytochemistry were obtained by withholding the primary antibodies and resulted in no staining .Neural crest stem cells and sensory neurons were cultured in 96-well plates and fixed with 4% paraformaldehyde, permeabilized with Triton X-100 (0.3%) and blocked with 3% fatty acid-free bovine serum albumin (BSA) . Cells were incubated for 2 h with primary antibodies diluted in 3% BSA. After washing with PBS, fluorescently labeled secondary antibodies were added for 40 min in the dark, washed thoroughly with PBS followed by a 5-min incubation with DAPI  for nuclear staining. After rinsing with PBS and water, 50 μl of glycerol were added as mounting medium in each well and the plates were sealed with aluminum sticker before analysis. The primary antibodies used were directed against: nestin , β-tubulin Class III , Islet1 , TRPV1 , BRN3A  and peripherin . Secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 . Images were acquired with a High-Content Screening microscope, Operetta  using a high numerical aperture 20x objective lens and specific filters for fluorescence excitation and emission. Image analyses were performed using high-content image analysis software Harmony 5.1  for morphology analysis and fluorescence quantification. Briefly, for quantification of immunostaining and cell counting, cells were segmented by proprietary algorithm  in a constitutive marker channel . The segmented areas in each image constituted a template used to measure fluorescence in the same image but in the channels related to the staining of interest. Three independent experiments were performed for immunofluorescence imaging and quantifications. Each marker was probed in triplicate in each experiment and twenty different unbiased microscopic fields were imaged in each replicate. Neurite length measurements were performed using proprietary algorithms as described above, however, TuJ1 staining was used as the primary marker for identifying, segmenting and measuring neuronal projections. The number of cell in each image was counted with DAPI staining and the ratio of TuJ1+ cells and DAPI stained nuclei was calculated. Fluorescence intensity measurements for Islet1 were performed using DAPI staining as the constitutive marker. Raw data of immunostaining quantifications are available on 2, 10 mM dextrose, 5 mM HEPES, 2 mM NaOH, pH 7.35, at room temperature (23°C). The microelectrode (intracellular) solution contained 120 mM K-gluconate, 45 mM KCl, 0.5 mM CaCl2, 5 mM EGTA, 10 mM HEPES, 2 mM ATP-Mg, 1 mM MgCl2, pH 7.35. Electrode resistance was 2.9–3.7 MΩ and corrections were applied for the estimated -13 mV liquid junction potential. Cells randomly selected for recordings had clear neuronal morphology, with 2–3 thin processes emerging from the cell body, a visible nucleolus and smooth surface under Hoffmann modulation contrast optics. Cells were first tested under current clamp for action potentials evoked by 500 ms depolarizing currents steps. Next, under voltage clamp, membrane potential was held at -70 mV and voltage-sensitive currents were evoked by 50 ms depolarizing pulses in 10 mV increments from -50 to +20 mV, preceded by a 20 ms hyperpolarization to -90 mV. Leak currents were subtracted with a P/4 protocol. Data were recorded and analyzed using WinWCP software, available at http://spider.science.strath.ac.uk/sipbs/software_ses.htm .Whole-cell patch clamp recordings were performed on neurons plated on PLO and laminin-treated coverslips, as previously described . The cov4, 1.5 CaCl2, 1 MgCl2, 10 D-glucose, 5 HEPES, pH 7.4.Neurons were plated onto PLO and Laminin-treated coverslips where they were treated with a HEKn CM for 10 days. On the day of the experiment, the medium was removed and cells were incubated with fresh medium (without factors) containing 5 μM Fura-2 AM  and 0.04% Pluronic acid for 1 h at 37°C. This solution was then replaced by fluorimetry buffer, composed of (in mM): 145 NaCl, 5 KCl, 1.2 NaHPOAfter 30 min at 37°C, the coverslips were placed in an Eclipse Ti inverted microscope  equipped with an Evolve 512 EMCCD camera , a Lambda DG-4 light source  and a fluid flow system. The images were acquired every 500 ms via MetaFluor software  and the levels of emitted fluorescence measured at 510 nm after stimulation with 340 and 380 nm were converted to a ratio (340/380 nm) and represented in pseudocolor. All drugs were freshly prepared from stocks and diluted in the fluorimetry buffer, with the control stimulation containing the vehicle (0.1% ethanol).4, 1.5 CaCl2, 1 MgCl2, 10 D-glucose, 5 HEPES, pH 7.4. Following washing, the cells were incubated with different concentrations of TRPV1 agonists, other agents, or 45 min in the same buffer. When used, antagonists were applied 15 min before the agonist addition. Subsequently, the supernatants were collected and immediately assayed with the ELISA kit, following the manufacturer’s instructions. All experiments were performed in triplicate and absorbance levels were measured in a microplate reader.Substance P (SP) levels were determined using a commercially available ELISA kit , following the manufacturer’s instructions. Sensory neurons were plated in 24-well plates at high confluence. After 24 h, cells were rinsed with Hanks’ balanced salt solution (HBSS) buffer, composed of (in mM): 145 NaCl, 5 KCl, 1.2 NaHPOAnandamide (AEA), rimonabant (SR141716A) and resiniferatoxin were obtained from Cayman Chemical, Co.  and hydrogen peroxide from Calbiochem.Supplementary Table 1).Total RNA from three independent cultures of hiPSC-derived neurons, matured in the absence (C) or presence of human epidermal keratinocyte conditioned medium (CM) were extracted using QIAGEN miRNAasy kit , following the manufacturer’s instructions. RNA concentrations were measured by spectrophotometry using Nanodrop . The integrity of the RNA was analyzed using Bioanalyzer  and the RNA integrity number (RIN) was obtained by identifying the ribosomal subunits in the samples. Only the samples with RIN greater than eight were sequenced, with an average coverage of more than 50 million reads per library. RNA sequencing was performed at Life Sciences Core Facility  using HiSeq 2500 platform (Illumina) in a paired-end mode . Cells were then dissociated, cultured in 96-well plates and characterized for the expression of specific markers. NCPCs were positive for Nestin , TRPV1 , Peripherin , BRN3A , Pax6  , merged images shown in Figures 2C,F,I,L,O. The expression of these markers was quantified as shown in Figure 2P. Similar results were also obtained with a different hiPSCs line  , and negFigure 1). Of note, neurons tended to form ganglion-like structures after 7 days in 3N medium, which is also described for mouse and rat primary cultures  and epidermal growth factor (EGF-) containing medium for 2 days. Subsequently, cells were plated onto Poly-Ornithine (PLO)/Laminin-coated plates. Cells were maintained in 3N medium with addition of brain derived neurotrophic factor (BDNF), AA, glial derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and cyclic AMP (cAMP), resulting in the formation of PSNs . The presence of HEKn CM accounts for a 17.4% increase in cell differentiation, when compared to control cultures maintained in regular 3N medium .Keratinocytes are important in the PSNs maturation process , thus, HFigure 4). Out of 10 cells from a single differentiation batch and sampled from different coverslips, nine fired at least one action potential under current clamp and all showed fast-inactivating inward currents under voltage clamp, typical of voltage-dependent sodium channels . Slowly-activating, non-inactivating potassium outward currents ranged from 0 to 1504 pA in response to a depolarizing step from -90 to 20 mV (mean: 611.2 ± 160.2 pA).Neuronal induction was further verified by electrophysiological recordings , Peripherin , and Islet1 . All markers showed increased expression over the observed time points . TRPV1 increase was the most noteworthy, enhancing approximately five times. These results suggest that CM promoted the maturation and growth of sensory neurons, as they exhibited higher expression of differentiation markers, hallmarks of the transition into sensory neurons maturity. The expression of all these markers, except Islet1, was also detected among RNA-Seq transcripts .There are several markers that make up the distinctive expression profile of somatosensory neurons. We assessed the expression of TRPV1 . Moreover, depolarization of neurons induced by a high potassium solution also showed weak calcium responses . These results suggest that in terms of intracellular calcium increases, these neurons have not fully matured.The TRPV1 channel is a non-selective cation-permeable channel, with significant calcium permeability . For thiFigure 6A). Interestingly, 100 μM anandamide evoked the highest SP release by the tested TRPV1 agonists with these cells. Moreover, when a high potassium solution (70 mM) was added to depolarize neurons, we obtained an increase in substance P release, though smaller then resiniferatoxin, bradykinin, and anandamide . In addition, the cells responded to 0.25% hydrogen peroxide by releasing the highest amount of SP.Since other authors have also observed very few calcium responses in hiPSCs-derived PSNs, attributing this lack of response to neurite activity rather than cell soma , we deciFigure 6B). Furthermore, AEA-increase of SP release was partially inhibited by capsazepine, a TRPV1-selective antagonist . Rimonabant, a CB1-selective antagonist did not block the AEA effect, on the contrary, it tended to increase it . These results indicate that functional nociceptive responses are present in these neurons, particularly TRPV1-mediated ones. The expression of the potential receptors involved in these functional responses are plotted in Figure 5E and include TRPV1, CB1 and CB2, and both bradykinin receptors, BDKRD1 and BDKRD2. Interestingly, CB2 was detected in negligible amounts.The anandamide effect, albeit at relatively high concentrations, was concentration-dependent in a range compatible with TRPV1 agonism rather than CB1 activation . Transcripts coding the same markers that were detected by immunostaining, such as Nestin (NES), Pax6 (PAX6), Peripherin (PRHP), BRN3A (POU4F1). TRPV1 was the most expressed TRP channel of sensory neurons, confirming the immunofluorescence staining. Also, the presence of the most expressed neuronal marker genes like PIEZO2 (piezo type mechanosensitive ion channel component 2), TH (tyrosine hydroxylase), TUBB3 (tubulin beta 3 class III), Neurotrophic receptor tyrosinase 2 , SCN3A and SCN3B  suggest that the obtained cell type has a C-low threshold mechanoreceptor (C-LTMR) profile, EGF , RUNX1T1 (RUNX1 translocation partner 1), CALCB (CGRP receptor component), LDHB (lactate dehydrogenase B), MDK , MSN (moesin) and P2RX3, P2RX4 (purinergic receptor P2X ligand-gated ion channel 3 and 4), were found at different levels in both PSN-C and PSN-CM transcriptional fingerprints, in three independent cultures per condition . RNA-Seq global expression data is available on Supplementary Datasheet 2 of the Supplementary Material.RNA-Seq was used to determine the transcriptional profile of the cells obtained using the differentiation protocol aforementioned. RNA from control PSN (PSN-C) and treated with HEKn-conditioned media (PSN-CM) during maturation in culture have both shown expression of all main expected neuronal markers  show about 10,325 genes significantly expressed (TPM > 10), with 9,610 genes expressed in both conditions, 266 expressed only in PSN-C and 449 genes exclusively expressed in PSN-CM condition, which could be interesting targets to be investigated to increase PSN maturation. Most of the transcripts that changed were downregulated after the treatment with HEK-CM . Moreover, mRNA encoding the cannabinoid receptor 1 (CNR1) was found in higher levels compared to TRPV1 . This could mean that the AEA-induced SP release via TRPV1 could be underestimated because CB1 is also stimulated by anandamide and might inhibit TRPV1. In fact, neurons pretreated with rimonabant, a CB1-selective antagonist, show a tendency to increase SP release with anandamide . Interleukin 1 α and β  transcripts were found only in PSN-CM cultures. Principal component analysis (PCA) was performed using ClustVis and provided an accurate verification of the applied method, as the first principal component clearly separated the samples by condition (C vs. CM) and the second principal component separated the samples by independent PSN cultures differentiation .Venn diagrams , suggesting that PSN present a transcriptional profile closer to neuronal than other human tissues or skin cells, like fibroblasts, which were the source of the cells that were reprogrammed to generate the hiPSC used in this study.The transcriptome comparison between PSN and GTEx RNA-Seq dataset from human tissues shows higher scaled correlation with brain tissues like hypothalamus, amygdala, frontal cortex, hippocampus  . For instance, CB1 (CNR1) is overexpressed  relatively to TRPV1. This overexpression could at least partially explain the low detectable activity of TRPV1, as CB1 is known to negatively modulate this channel through dephosphorylation   . AccordiIn addition, in the case of sensory neurons expressing TRPV1 channels, these proteins are packaged into CGRP- or SP- and VAMP1-containing vesicles and delivered to the plasma membrane, involving the formation of SNARE complexes composed of SNAP-25, syntaxin 1, and VAMP1, as well as Munc18–1 . This deSupplementary Figure 9)  . Again, We have shown that, although a negligible number of cells respond to capsaicin measured by intracellular calcium increases, we could detect substance P release under similar conditions. Release of SP or any other neuropeptide is thought to be dependent of intracellular calcium increases, which would allow the proper fusion of synaptic vesicles to the presynaptic membrane thereby releasing its contents in the synaptic cleft. These two findings would therefore seem irreconcilable. However, in vitro does not guarantee that this activity would be satisfactory in the end, as mentioned. Therefore, we must consider additional mechanisms to enrich the neuronal population in TRPV1+/TrKA+/CGRP+ cells. First, the amount of NGF necessary might be higher, or perhaps a gradient of this neurotrophin might be necessary. NGF is present in high concentrations within the epidermis, where nerve fibers with free endings  will project  in adult DRGs  subtype 3.2, uniquely expressed in unmyelinated C-LTMR . However, to precisely define the proportion of this neuronal type in the culture further investigations have to be performed using single cell RNA sequencing.Although individual cell gene expression variability is influenced by differentiation conditions, our PSN expressed the five sensory-neuronal genes marker genes, except SCN9A . TH, whiult DRGs  and was ult DRGs  as shownd C-LTMR . Togethed C-LTMR . Our RNAin vitro models using these differentiated human neurons.In summary, although some reports have described the generation of human PSNs, none have successfully demonstrated a robust and useful TRPV1 activation. The present work shows detectable TRPV1 activity, the release of SP mediated by resiniferatoxin and anandamide that could be used for screening of nociceptive agents and possibly analgesics. The transcriptional data obtained from RNA-Seq show the main neuronal marker genes expressed, in different levels, in independent PSN cultures. The strategy of treating PSN with CM to investigate whether it could increase cell maturity and TRPV1 functionality had opposed outcomes. Although neurite growth increased, which is an indication of neuronal maturity, there were no conclusive changes in relevant neuronal markers expression in RNA-Seq data, although TRPV1 immunostaining increased. Nevertheless, as TRPV1 activity would be useful for the purpose of screening for new nociceptive/irritant agents, it is appealing to further improve the robustness of this activity and to develop reliable The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.MG, RDV, JS, BP, RdC, NC, LB, and SR conceived and designed the experiments. MG, RDV, GV, JS, BP, IL, RdC, and NC performed the experiments. MG, RDV, BP, RdC, FRdS, and NC analyzed the data. MG, RDV, BP, RdC, FRdS, NC, LB, and SR wrote the paper.LB and RDV are employees of L’Oréal Research & Innovation. The remaining authors have received sponsored research support from L’Oréal Research & Innovation.