Cucumis sativus L.) is one of the most economically important fruits of the Cucurbitaceae family, therefore consideration of potential pesticide residues in the fruit in the context of cucumber breeding and production programs is important. Propamocarb (a pesticide commonly used to prevent downy mildew) is widely used in cucumber cultivation, but the molecular mechanism underlying the degradation and metabolism of propamocarb in cucumber is not well understood. We screened a candidate CsHMGB gene (CsaV3-5G28190) for response to propamocarb exposure using transcriptome data. The coding region of CsHMGB was 624 bp in length and encoded the conserved HMB-box region. CsHMGB expression differed significantly between the “D0351” genotype, which accumulated low levels of propamocarb, and the “D9320” genotype, which accumulated high levels of propamocarb. CsHMGB expression was positively correlated with propamocarb levels in the cucumber peel. CsHMGB expression was upregulated in the fruit peels of the “D0351” genotype following exposure to propamocarb stress for 3–120 h, but no difference was observed in expression between propamocarb treatment and control for the “D9320” genotype. For the “D0351” genotype, CsHMGB expression was higher in the fruit peels and leaves than that in female flowers; expression was moderate in the stems and fruit pulps, and weak in male flowers and roots. The CsHMGB protein was targeted to the nucleus in Arabidopsis protoplasts and in the epidermis of Nicotiana benthamiana leaves. We measured MDA, O2–, and H2O2 levels in cucumber plants and found that they were likely to accumulate reactive oxygen species (ROS) in response to propamocarb stress. Analysis of antioxidant enzyme activity  and the ascorbate-glutathione (AsA-GSH) system showed that the resistance of the plants was reduced and the levels of propamocarb residue was increased in CsHMGB-silenced plants in response to propamocarb stress. Conversely, overexpression of CsHMGB promoted glutathione-dependent detoxification by AsA-GSH system and improved the antioxidant potential, reduced the accumulation of ROS. Ultimately, the metabolism of propamocarb in cucumber was increased via increase in the wax levels and the stomatal conductance.Cucumber ( Cucumis sativus L.) is one of the most economically important fruits in the Cucurbitaceae family and is widely cultivated in greenhouses, where it is susceptible to downy mildew (Cucumber (y mildew . Propamoy mildew . Most pey mildew . ExcessiCsHMGB) which was upregulated in response to propamocarb stress  protein is a highly abundant non-histone chromosomal protein that is found in eukaryotes. Human diseases, cell damage, trauma-induced stress and necrosis, chronic inflammation, autoimmune diseases, and cancer may all lead to the release of the HMGB1 protein. Extracellular HMGB1 stimulates the inflammatory response and recruits leukocytes by interacting with receptors for advanced glycation end-products (RAGE), toll-like receptor 2 (TLR), and other receptors to protect the organism from disease . In pland stress . AtHMGB1t stress . AtHMGB1len tube . AtHMGB3callosum . The HMGcallosum . HoweverS-transferase (GST) and glycosyltransferase; (3) transmembrane transport of transporters such as the ABC family; and (4) the hydrolase degradation reaction. The first two steps degrade toxic macromolecular groups into lower levels of toxicity and create poor mobility metabolites, and the latter two steps are responsible for the further hydrolysis and transport of low-toxicity small molecular compounds ] in plants can be divided into four phases: (1) hydrolysis of peroxidase and cytochrome P450s; (2) catalysis of glutathione-ompounds . Additioompounds . Pesticiompounds . Wax is ompounds .CsHMGB, involved in mediating propamocarb stress. Functional analyses revealed that overexpression of CsHMGB could alleviate propamocarb phytotoxicity by increasing the waxiness of the cuticle, increasing the stomatal conductance in the pericarp cells, and activating the antioxidant system.To investigate the mechanisms underlying propamocarb residue accumulation in cucumbers, we identified a putative gene, Two cucumber genotypes exhibiting different levels of propamocarb residues in the fruit peel were used: “D0351”  and “D9320” . For each genotype, 200 plants were sown in pots containing a mixture of soil and vermiculite (v:v = 1:1) in a solar greenhouse at the Northeast Agricultural University  . Plants To investigate phytotoxicity in cucumber plants, we sprayed 20 μL propamocarb  onto the leaves of “D0351” and “D9320” plants. Leaves of similar size were selected at 48 h after propamocarb application for further study. MDA content was measured by reacting MDA with thiobarbituric acid (TBA), and MDA-TBA adduct formation was measured based on absorbance at 450, 532, and 600 nm wavelengths .To determine the accumulation of propamocarb residues in various organs, we applied 50 mL propamocarb (400 ppm) to the entire plant until liquid dripped from the leaves and fruits at 34 days after planting; an equivalent volume of water was applied as a control. Each plant was sprayed once. Samples were collected from five individual plants at 6, 9, 12, 24, 36, 48, 72, 96, and 120 h after propamocarb application. Fruit were collected from the 10th node. Fruits with the following parameters were selected: length 16–18 cm, diameter 5–6 cm, and weight 160–180 g . Fruit pThe “D0351” genotype was used for the gene silencing experiment and was planted in an artificial climate incubator .1 lines of OX2, OX3, and OX5  were sown in growth chambers. Plants were treated with propamocarb, and the samples were harvested as previously described.The “D9320” genotype was utilized for overexpression studies. T® (CAS: 24579-73-5) . Ten grams of homogenized sample were added to a 50 mL colorimetric tube and further homogenized for 5 min. Then, 20 mL acetonitrile and 2.5 g NaCl were added to the sample in the 50 mL colorimetric tube, and mixed for 5 min with shaking at 500 g/min using a mechanical shaker. Samples were allowed to settle for 30 min and then vortex mixed and evaporated until dryness; this step was repeated several times and the resulting material was collected. The sample was then filtered through a polypropylene filter  (0.22 μm) until no residual particles remained. The levels of propamocarb residue were then analyzed using the Agilent 7890B-5977A gas chromatography system (Agilent Technologies) with a 30 m × 0.25 mm × 0.25 μm capillary column (HP-5MS). The oven temperature was programmed at 40°C 2 min, increased to 200°C at 25°C/min and held for 8 min, sample inlet: 240°C, non-split injection, flow rate of 1.0 mL min–1.A propamocarb analytical standard was provided by Sigma-AldrichCsHMGB gene was acquired from the cucumber genome database1. Fruit peels from cucumber genotype “D9320” were used to clone CsHMGB with specific primers  was used to construct a neighbor-joining (NJ) phylogenetic tree (The coding sequence for the full-length  primers . Polymertic tree .–1), and ddH2O. Cucumber EF1a was used as an internal reference gene to calculate the relative expression of the candidate genes . This RNA was then used to synthesize single-stranded cDNA using a reverse transcription kit , And qRT-PCR was performed in a 20 μL reaction containing 2× Fast qPCR Master Mix , cDNA template (2 μL), forward and reverse primers  was amplified using PCR. CsHMGB was cloned into a green fluorescent protein (GFP) carrier with a super promoter (pSuper-1300) via traditional digestion (XbaI and BglII) and T4 ligation   . The empd 580 nm   suppresses the silencing of CsHMGB . A specific 248 bp fragment of CsHMGB was amplified and then cloned into the pTRV2 vector using EcorI with BamHI. Plasmids were used to transform A. tumefaciens strain GV3101, which were then cultivated in LB medium containing 50 μg mL–1 rifampicin and 50 μg mL–1 kanamycin on a rocking platform at 200 rpm and at 28°C for 18 h. Cells were collected and re-suspended in an infiltration buffer supplemented with 200 mM acetosyringone, 10 mM MgCl2, and 10 mM MES (pH 5.6), at a final OD600 of approximately 1.2. Culture mixtures primarily contained pTRV2-CsHMGB and pTRV1-CsHMGB , or pTRV2 and pTRV1 (negative controls), and were incubated for 3–4 h under 25°C in darkness prior to injection. Silencing of the plant alkene desaturase (PDS) gene causes photobleaching, which can be used to control the efficiency of VIGS (Virus-induced gene silencing (VIGS) for  of VIGS . In totaCsHMGB sequence was cloned into the pCXSN-1250 vector harboring the glyphosate resistance gene under a constitutive promoter , thus generating the pCXSN-1250-CsHMGB overexpression vector. Agrobacteria from the GV3101-mediated method were used to infect the cotyledons of cucumber plants to generate transgenic plants. Cells with over-expression were selected in MS medium supplemented with 1 mg/L glufosinate , immersed in the solution by vacuum pumping, and stored at 4°C for 1.5 h. Samples were then flushed, dehydrated, replaced, and freeze-dried. Conductive tape was glued to the scanning electron microscope table, and the sample surface was coated with a 15 nm metal film using an Emur1010 (HITACHI) ion sputtering coating instrument. Samples were observed and imaged under a Smur3400N scanning electron microscope.Samples were collected from five individual plants at 2, 4, 6, 8, and 10 days after propamocarb application . Leaves 2O2 content was measured by sulfuric acid peptide oxidation at a 415 nm characteristic absorption. The 0.5 g sample was grinded in the 2 mL pre-cooled acetone in an ice bath followed by centrifugation for 20 min at 10,000 g at 4°C, the supernatant was brought to 3 mL. Then, 1 mL extracting solution, 0.1 mL 20% titanic tetrachloride and 0.2 mL concentrated NH4OH was mixed for 5 min and centrifuged for 15 min at 10,000 g at 4°C. The supernatant was discarded and the precipitate was dissolved in 1 mol L–1 H2SO4, and washed several times with cold acetone, the final volume was made up to 2 mL  with full grinding, and collecting the supernatant after centrifugation at 10,000 g for 20 min, then the volume was adjusted to 3 mL. The 0.5 mL extracting solution mixed with 0.5 mL phosphoric acid buffer  and 1.5 mL hydroxylamine hydrochloride (1 mol L–1), placed at 25°C for 1 h. Then, addition with 2 mL p-aminobenzene sulfonic acid (17 mol L–1) and 2 mL α-naphthylamine (7 mol L–1), the mixture was incubated at 25°C for 20 min  in an ice bath followed by centrifugation for 20 min at 10,000 g at 4°C. Extracting solution was used to determine the SOD, POD, CAT, and APX activity. POD activity was determined via the catalytic H2O2 oxidation of specific substrates by the guaiacol method at 470 nm . The absorbance at 290 nm was measured at every 15 s intervals for 3 min . The absorbance at 290 nm was measured at 10 and 130 s; DHA content was calculated from the rate of AsA formation at a wavelength of 265 nm at 10 and 130 s .The oxidation rate of ASA measured at 265 nm was used to estimate ASA content. The 0.5 g sample was ground to a homogenate with 5 mL buffer I in an ice bath followed by centrifugation for 20 min at 8,000 –1 phosphoric acid buffer  in an ice bath followed by centrifugation for 20 min at 16,000 g at 4°C. A total of 0.3 mL extracting solution with 50 mol L–1 phosphoric acid buffer (pH = 7.0), 20 mol L–1 GSH and 2 mmol L–1 DHA up to 3 mL was used to measure DHAR activity at 265 nm. A total of 0.09 mL extracting solution with 50 mol L–1 phosphoric acid buffer , 2 mmol L–1 NADPH and 2 U AAO (pH = 5.6) was used to measure MDHAR activity at 340 nm.DHAR and MDHAR activity were analyzed as described by –1 PBS, 0.2 mol L–1 NADPH and 1 U GR up to 3 mL. The mixture was incubated at 27°C for 30 min and the absorbance was at 412 nm measured for total glutathione content. The difference between the determination of GSSG and total glutathione was the reaction system containing 0.1 mol L–1 2-vinylpyridine, and the mixture was incubated at 27°C for 1 h for determination of GSSG content. The GSH content was then calculated by subtracting GSSG from total glutathione.Total glutathione and GSSG content were analyzed as described by S-transferase activity was measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) and 5 mM GSH as standard substrates at a wavelength of 340 nm. The 0.5 g sample was ground to a homogenate with 5 mL 50 mol L–1 phosphoric acid buffer  and polyvinyl-polypyrrolidone (insoluble PVPP), followed by centrifugation for 20 min at 12,000 g at 4°C , the volume was adjusted to 4 mL followed by centrifugation for 15 min at 16,000 g at 4°C. A total of 0.1 mL supernatant was mixed with 0.1 mM Tris-HCl (PH = 8.0), 1 mol L–1 GSSG and 0.5 mM NADPH up to 3 mL with a characteristic absorption peak at 340 nm (GR activity was detected via the estimation of the dehydrogenation rate of NADPH. The 0.5 g sample was grinded in the reaction mixture (0.1 mol Lt 340 nm .t tests (p < 0.05 significance level and p < 0.001 extremely significant level).The statistical significance of differences was calculated by Tukey’s tests using Statistical Analysis System (SAS) version 9.21. Data are means (±SE) of three independent experiments each corresponding to at least three replicates. Statistically significant differences were determined by Student’s To determine the phytotoxicity of propamocarb in cucumber, we applied three different concentrations of propamocarb  to the leaves of the “D0351” and “D9320” genotypes. The recommended dose of 400 ppm did not induce phenotypic changes in cucumber. However, excessive use of propamocarb  could induce phytotoxicity, such as yellow speckles on leaves . MDA conWe detected the propamocarb residues in “D0351” and “D9320” genotype, results showed that the maximum residue in found in the fruit peels was 2.08-fold higher in “D9320” than that in “D0351” after 2 days of treatment with 400 ppm propamocarb. Dramatic changes in fruit peel propamocarb residue were noted in both “D0351” and “D9320” after application for 3 h to 21 days. Propamocarb residues gradually increased from 12 h to 2 days of propamocarb application and dramatically decreased from 2 to 5 days. However, propamocarb residues rarely decreased between 5 and 21 days of propamocarb application . DetectiCsaV3-5G28190 expression significantly changed under propamocarb stress, and the calculated transcript level was consistent with qRT-PCR results. The full-length CsHMGB cDNA sequence was cloned through RT-PCR based on the “D0351” genotype total RNA. That genomic coding region was 624 bp in length, including eight exons and seven introns with the start codon of ATG to TAA. The amino acid sequence of CsHMGB was obtained from cucumber plants, HMGB proteins were obtained from other Cucurbitaceae crops, and HMGB1/2/7 was obtained from Arabidopsis and other plant species. The results indicated that CsHMGB from cucumber, CmHMGB7 from melon (Cucumis melo L.), MnHMGB7 from bitter gourd (Momordica charantia L.), and CpHMGB7 from pepper (Capsicum annuum L.) were in the same evolutionary branch, as their genetic relationships were greater than 80%. The alignment of amino acid sequences showed that the CsHMGB protein contained the HMB-box conserved region . In CsHMGB-silenced plants, O2– and H2O2 contents increased by 127 and 125%, respectively, in comparison with the control after 48 h of propamocarb stress  system, such as DHAR . These changes suggested that silencing of CsHMGB promoted the conversion of ascorbate from AsA to DHA, leading to the decreased of the redox state of the ascorbate. Total glutathione content did not differ between the control and the CsHMGB-silenced plants; however, the ratio of GSH/GSSG was lower in the CsHMGB-silenced plants than in the control, indicating that the cucumber plants accelerated GSH consumption in response to propamocarb stress  were identified by qPCR through genetic transformation. Three OE lines, which shared the most abundant contents of CsHMGB , were harvested from the T1 generation after self-pollination (The overexpressed-vector of lination .CsHMGB-overexpressed plants and the control plants. We observed no phenotypic differences (such as fruit length and color of fruit peel) between the CsHMGB-overexpressed plants and the control plants  proteins were identified in cucumbers, and cluster analysis showed that the HMGB proteins were contained in one branch between cucumber and A. thaliana (CsHMGB (CsaV3_5G021890.1) shared the highest sequence similarity with AtHMGB5 (AT4G35570.1) was screened for gene function under propamocarb stress. We found that CsHMGB was localized to the nucleus  and exogenous melatonin alleviated carbendazim phytotoxicity and pesticide residue levels by improving the activity of antioxidant enzymes and activation of the AsA-GSH system and reduction of ROS and MDA levels (RBOH1) resulted in H2O2-dependent redox homeostasis, glutathione biosynthesis, and increased GST activity to limit CHT residue levels in tomatoes  and the AsA-GSH system were also observed. Overexpression of CsHMGB promoted glutathione-dependent detoxification by AsA-GSH system and improved the antioxidant potential, reduced the accumulation of ROS. Ultimately, propamocarb metabolism in cucumber was increased via increase in the wax levels and the stomatal conductance (CsHMGB contributes to lower propamocarb residue levels, with possible applications in the production and breeding of cucumbers to achieve lower propamocarb residue levels in fruits.We cloned a 624 bp of ductance . Thus, Chttp://www.icugi.org/.The datasets generated for this study can be found in the SL and ZQ designed and conceived the research. MX, JL, CW, DL, WZ, and XZ performed the experiments. CW, DL, CL, and WZ analyzed the sequencing data. SL wrote the entire manuscript. ZQ and XZ edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that symmetrically di-methylates arginine residues on both histone and non-histone protein substrates. Accumulating evidence suggests that PRMT5 exerts its oncogenic properties in a wide spectrum of human malignancies. However, the underlying mechanisms by which PRMT5 contributes to the progression of colorectal cancer (CRC) remain to be defined.Methods: Western blot and real-time PCR were used to analyze the expression of CDKN2B. Co-immunoprecipitation (Co-IP), immunofluorescence and GST pulldown assays were employed to investigate the interaction between PRMT5 and EZH2. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to validate CDKN2B as a direct target of PRMT5/EZH2. DNA methylation status at the CpG islands of promoter region of CDKN2B gene was analyzed by bisulfite sequencing. The effect of PRMT5/EZH2 on malignant phenotypes was examined through in vitro and in vivo assays. PRMT5 and EZH2 protein expression levels in CRC tissues were analyzed by immunohistochemistry (IHC) staining.Results: We observed that PRMT5-deficient CRC cells exhibit proliferation defects in vitro. PRMT5 was identified as a major transcriptional repressor of CDKN2B (p15INK4b) for determining CRC progression. Mechanistically, PRMT5-mediated histone marks H4R3me2s and H3R8me2s were predominantly deposited at the promoter region of CDKN2B gene in CRC cells. Knockdown of PRMT5 in CRC cells decreased the accumulation of H4R3me2s and H3R8me2s marks and reduced the CpG methylation level of CDKN2B promoter, then re-activated CDKN2B expression. Strikingly, silencing of CDKN2B partially abrogated the proliferation defects caused by PRMT5 depletion in vitro and in vivo. Furthermore, we proved that PRMT5 interacted with Enhancer of zeste homolog 2 (EZH2), leading to enhanced EZH2 binding and H3K27me3 deposition together with decreased transcriptional output of CDKN2B gene. Importantly, we found that the combined interventions exerted a synergistic inhibitory effect of combined treatment with PRMT5i (GSK591) and EZH2i (GSK126) on the growth of CRC cells/xenografts in vitro and in vivo. Moreover, PRMT5 and EZH2 were found to be significantly elevated and associated with poor prognosis in CRC patients.Conclusion: PRMT5 functionally associates with EZH2 to promote CRC progression through epigenetically repressing CDKN2B expression. Thus, our findings raise the possibility that combinational intervention of PRMT5 and EZH2 may be a promising strategy for CRC therapy. Colorectal cancer (CRC) is the third most common diagnosed cancer in the world G-monomethylarginine (MMA), ω-NG, NG-asymmetric dimethylarginine (ADMA), and ω-NG, NG-symmetric dimethylarginine (SDMA) Arginine (R) methylation is emerging as an important post-translational modification (PTM) involved in a wide spectrum of intracellular events, including pre-mRNA splicing, gene transcription and mRNA translation in vitro, as well as tumor growth in vivoEnhancer of zeste homolog 2 (EZH2) is a catalytic component of Polycomb repressive complex 2 (PRC2) that specially catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) by SET domain in its C-terminus PRC2 consists of four core subunits, EZH2, SUZ12 (suppressor of zeste 12 homolog), and EED (embryonic ectoderm development), and RBBP4/7 (retinoblastoma binding protein 4/7), and is involved in transcriptional silencing of growth and development-related genes in vitro and tumor growth in vivo. Mechanistic studies revealed that PRMT5 epigenetically represses the transcription of CDKN2B (p15INK4b) gene through association with EZH2 in CRC cells. Further analysis showed that the combined interventions exerted a synergistic inhibitory effect of combined treatment with PRMT5i (GSK591) and EZH2i (GSK126) on the growth of HCT116 cells/xenografts in vitro and in vivo. Our data support the concept that combinational inhibition of PRMT5 and EZH2 may be a promising strategy for CRC therapy.In the present study, we report that PRMT5 facilitates the cell proliferation 2.Human CRC cell lines (HCT116 and SW480) and human embryonic kidney cell line (HEK-293T) were obtained from the Cell Bank of the Chinese Academy of Sciences . HCT116 (poorly differentiated) and NCM460 (noncancerous colon) cells were culture in McCoy's 5A medium . SW480 (moderately differentiated) and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium . All culture media were supplemented with 10% fetal bovine serum  and 1% antibiotics . CRC cells were incubated at 37 °C in a humidified incubator with an atmosphere of 5% COShort hairpin RNAs (shRNAs) as well as a control shRNA were designed to silence PRMT5, EZH2 or CDKN2B. Control shRNA: 5'-TTCTCCGAACGTGT CACGT-3'; shPRMT5-1: 5'-GGGACTGGAATACGCTAATTG-3'; shPRMT5-2: 5'-GGAATCTCAGACATATGAAGT-3'; shEZH2-1: 5'-GCTAGGTTAATTGGGACCA AA-3'; shEZH2-2: 5'-CCAACACAAGTCATCCCATTA-3'; shCDKN2B: 5'-GGTGC GACAGCTCCTGGAA-3'. DNA sequences coding corresponding shRNAs were synthesized and then inserted into the lentiviral plasmid pLKO.1-TRC. psPAX2 (packaging plasmid) and pMD2.G (enveloping plasmid) and pLKO.1-TRC-shRNA were transiently transfected into HEK-293T cells using Lipofectamin 2000 (Life Technologies) to prepare the lentiviruses. 48 ~ 72 hours later after transfection, culture supernatants were harvested and filtered through 0.22 μm-pore-sized filters . For virus infection, HCT116, SW480 or NCM460 cells were treated with harvested viral supernatants. Polybrene  was added to the cultures to improve the infection efficiency as an enhancing reagent. 48 ~ 72 hours later, infected HCT116, SW480 or NCM460 cells were selected with puromycin  for 5 ~ 7 days.4 cells/mL (100 µL/well) and incubated at 37 °C with 5% CO2. The optical density of the solution (OD450) in each well was measured by spectrophotometry (BioTek) on days 0, 1, 2 and 3 for HCT116 and SW480 cell, following addition of 10 µL CCK-8 reagent.A CCK-8 Cell Counting Kit  was used to assess the proliferation of HCT116 and SW480 cells. Cells were seeded in 96-well plates at a density of 2×104 cells per well in a 24-well plates and incubated at 37 °C with 5% CO2 overnight. The EdU incorporation assay was carried out according to the instructions for the BeyoClick™ EdU-488 assay kit . After incubation with 10 μM EdU for 2 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and stained with fluorescent dyes (green). 1 × Hoechst 33342 was used to stain the cell nuclei  at room temperature for 10 min. Cells were observed under a fluorescent microscope (Nikon).For EdU (5-ethynyl-2'-deoxyuridine) assay, cells were seeded in 96-well plates at a density of 5 × 103 cells per well in 6-well plates and then cultured in medium supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C with 5% CO2. After macro-clones (>50 cells/colony) formed, cells were fixed with 100% methanol and stained with 0.1% (m/v) crystal violet solution at room temperature.Plate colony-formation assay was performed to determine the colony-formation capabilities of HCT116 and SW480 cells. Cells were seeded at 1×10-ΔΔCt method was used to calculate relative mRNA amounts of target genes to the endogenous GAPDH control. The sequences of real-time PCR primers were listed in Total RNA was extracted using Trizol reagent  and the ratio of A260/A280 was examined by a NanoDrop 2000 spectrophotometer (Thermo Scientific). A total of 0.5 ~ 1 µg of purified RNA was reversed-transcribed to cDNA with the BeyoRT™ II First Strand cDNA Synthesis Kit with gDNA Eraser  in the following conditions: 65 °C for 5 min, 42 °C for 60 min and 80 °C for 10 min, and then subjected to real-time PCR analysis. Real-time PCR was performed using the BeyoFast™ SYBR Green qPCR Mix  on an ABI-7900 instrument (Applied Biosystems). The real-time PCR program steps were as follows: 95 °C for 5 min, then 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The 2Whole-cell lysates of HCT116 and SW480 cells were extracted using lysis buffer, including 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were centrifuged at a speed of 14000 rpm for 15 min, then mixed with 5×loading buffer and denatured at 100 °C for 5 min. Aliquots of supernatant protein (40 ~ 50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The PVDF membranes were then blocked for 1 h with 5% (w/v) non-fat milk at room temperature. After incubation with anti-PRMT5 , anti-EZH2 , anti-CDKN2B  or anti-GAPDH  overnight at 4°C, the PVDF membranes were washed with PBST and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich) at room temperature. Subsequently, the immunoreactive bands were visualized using ECL detection reagent (Thermo Scientific).E. coli strain BL21 (DE3) transformed with pGEX plasmids encoding GST or GST-PRMT5 fusion protein. Briefly, the plasmids were transformed into E. coli strain BL21 (DE3), and then cultured and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) to express GST or GST-PRMT5 fusion proteins. The GST recombinant proteins were purified with glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) from the lysate of E. coli strain BL21 (DE3). Similarly, His-tagged EZH2 fusion protein was expressed in E. coli strain BL21 (DE3) and the His-tagged EZH2 fusion protein was purified with Ni-NTA agarose beads (Qiagen). GST-tagged PRMT5 or His-tagged EZH2 recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue to visualize His or GST fusion proteins. Subsequently, GST pulldown assays were performed to validate the direct interaction of GST-tagged PRMT5 and His-tagged EZH2 in vitro. Equal amount of GST or GST-tagged PRMT5 fusion proteins were incubated with His-tagged EZH2. After washing, the bound fusion proteins were eluted from glutathione Sepharose 4B beads, separated by SDS-PAGE and detected by immunoblotting with anti-EZH2 antibody.Glutathione S-transferase (GST) recombinant proteins were expressed in in vitro. To obtain promoter-luciferase reporter constructs, promoter region of the human CDKN2B gene (-2000 to -1 bp) was PCR-amplified from genomic DNA, cloned into the upstream of the luciferase reporter gene in the promoter-less pGL3.0-empty vector (Promega) and confirmed by DNA sequencing. Next, these constructs and PRMT5-overexpressing plasmid (pCMV-PRMT5) were co-transfected into HCT116 and SW480 cells using Lipofectamin 2000 (Life Technologies). Transiently transfected HCT116 and SW480 cells were cultured for 48 ~ 72 hours and then lysed with lysis buffer . The luciferase activity was assessed using the dual luciferase reporter assay system (Promega) according to the manufacturer's protocol.Luciferase reporter gene assays were used to monitor the transcriptional activity of specific genes Cells were washed with 1 × PBS, and then the genomic DNA was extracted from the cells using the DNeasy Tissue Kit (Qiagen). DNA methylation status at the CpG islands of promoter region of CDKN2B gene was analyzed by bisulfite sequencing (BS). Bisulfite conversion of genomic DNA was performed using the EZ DNA Methylation-Gold kit (Zymo Research) according to manufacturer's instructions. The bisulfite-treated genomic DNA were subjected to PCR for the amplification of the CpG islands of CDKN2B promoter using Hot Start Takara Taq DNA Polymerase (Takara). Amplified PCR products were cloned into the pGEMT-easy vector (Promega) using DNA Ligation Kit (Takara). Ten insert-positive clones were isolated by the QIAprep Spin Miniprep kit (Qiagen) and sequenced using the ABI sequencing system (Applied Biosystems). The PCR primers used for bisulfite sequencing of CpG islands of promoter region of CDKN2B gene are presented in Cells were fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 30 min and permeabilized with Triton X-100 for 40 min at room temperature. Primary antibodies for staining PRMT5  and EZH2  were used at 1:200 and 1:500 for 1 h at room temperature, respectively. After washing with HEPES buffered saline , cells were incubated with secondary antibodies (Alexa Fluor-488 anti-mouse and Alexa Fluor-546 anti-rabbit) for 40 min. After washing with PBS, cells were stained with 4, 6-diamidino-2-phenylindole dihydrochloride  for 5 ~ 10 min. Stained cells were mounted with anti-fade mounting medium (ProLong Gold Antifade), covered with coverslips and visualized with epifluorescence microscope (Nikon).https://www.oncomine.org) according to the following criteria: (I) gene: PRMT5, EZH2 and CDKN2B; (II) cancer type: colorectal cancer; (III) data type: all; (IV) analysis type: cancer analysis vs. normal analysis; (V) thresholds: P value <1E-4, fold change >2 and gene rank = top 10%.The available data was assessed using the Oncomine database  to block endogenous peroxidase activity and to avoid immunoreaction. Subsequently, the slides were incubated with the primary antibodies more than 4 hours, including PRMT5 , EZH2  and CDKN2B . After washing with PBS, the sections were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase  and 3, 3'-diaminobenzidine  Substrate solution to visualize the immunoreaction. The staining of CRC slides were reviewed by two trained pathologists blindly and independently. Semi-quantitative of protein expression was calculated according to staining intensity and percentage of positive cells to generate a histological score . H score = ΣPi (i + 1), where is the intensity of staining (0 ~ 3) and Pi is the percentage of staining positive cells (0% ~ 100%).This study was approved by the Ethics Committee of the The Affiliated Cancer Hospital of Nanjing Medical University, while informed consents were provided by all the patients. Surgically derived CRC biopsies were fixed in formalin solutions , embedded in paraffin and sectioned at 3 ~ 5 μm thickness. The slides were washed with 3% hydrogen peroxide  and the chromatin was sonicated to generate 200 ~ 500 bp DNA fragments. Immunoprecipitation was performed by adding either anti-PRMT5 , anti-H4R3me2s , anti-H3R8me2s , anti-EZH2 , H3K27me3 , anti-IgG  and Protein G agarose beads  to each sample and incubated overnight at 4 °C. Following extensive washing with, the complexes were decrosslinked at 65 °C and treated with Proteinase K (Roche). The eluted DNA was precipitated with ethanol at -20 °C in the presence of Glycogen  as a carrier, washed twice with 70% ethanol, and then subjected to real-time PCR using primers specific to the target gene promoters, which were listed in The combination therapies were performed in 96-well plates. Cells were treated with PRMT5 inhibitor-GSK591 , EZH2 inhibitor-GSK126 , or the combination at graded concentrations for 7 days, and cell proliferation was determined by CCK-8 assays. To determine the presence of a possible synergistic effect of PRMT5i (GSK591) and EZH2i (GSK126), the combination index (CI) was calculated using CompuSyn software. A CI < 0.8 indicates synergy and CI > 1.0 indicates an antagonistic effect.in vivo experiments were purchased from the Experimental Animal Center, Chinese Academy of Sciences . All the nude mice were housed under a standardized light-dark cycle (12:12) in standard cages and allowed to free access to food and water. The experimental protocol and all animal experimental procedures were approved by the Animal Care and Use Committee of the Affiliated Cancer Hospital of Nanjing Medical University. Cells were suspended in FBS-free McCOY's 5A medium at a concentration of 2×107 cells/mL, and then implanted subcutaneously into the mouse right flank of each mouse (2×106 cells per mouse). The mice were followed for the observation of xenograft growth and measured by manual caliper every three days.Six-week-old female nude mice  used for the in vivo drug studies, tumor-bearing mice were randomized into four groups (7 mice per group), and drug dosing was initiated when average tumor volume reached 50 mm3 (at day 4). PRMT5i  and EZH2i  were dissolved in 20% Sulfobutylether-β-Cyclodextrin . GSK591 and GSK126 were administered intraperitoneally every other day at a dose of 100 mg/kg for a month. Tumor growth was monitored by the measurement of subcutaneous tumor volume as above. Mouse body weight was also measured by manual caliper every four days. The tumor volumes were calculated as 0.5 × length × width2. At the end point, mice were sacrificed, and xenografts were surgically isolated, weighted and photographed. Tumor growth inhibition (TGI) rate was calculated as shown: TGI = [1 - (tumor volume of control on day 36 - tumor volume of treated on day 4) / (tumor volume of control on day 36 - tumor volume of control on day 4)] × 100.For t-test to evaluate the significance of the differences between two groups. To analyze the relationship between positive PRMT5/EZH2 expression and clinicopathological parameters of CRC patients, student's t-test was used to compare values for the two parameters in each category. Survival was assessed by the Kaplan-Meier method and the survival curves were compared using the log-rank test. Data were reported as the mean ± SD from three independent experiments. P values < 0.05 (two-tailed) were considered statistically significant. * and ** are denoted as P < 0.05 and P < 0.01, respectively.All statistical analysis was performed using GraphPad Prism 7.0 . Statistical analyses were performed using two-tailed Student's P < 0.01). Marked decreases in colony-forming abilities were also evident in PRMT5-deficient HCT116 and SW480 cells . Ethynyl-2'-deoxyuridine (EdU) incorporation is an effective and precise way to evaluate cell proliferation in vitro. As shown in Figure P < 0.01), we observed that shRNA-mediated depletion of PRMT5 greatly inhibited EdU incorporation in HCT116 and SW480 cells. These findings together suggest that PRMT5 is associated with a highly proliferative tumor phenotype in CRC cells, emphasizing the critical role of PRMT5 in regulating CRC progression.To understand the contributing roles of PRMT5 on CRC progression, we firstly evaluated the effects of lentivirus-mediated shRNA targeting PRMT5 on cell proliferation of human CRC cell lines-HCT116 and SW480. We observed that the expression levels of PRMT5 in shPRMT5 lentivirus-infected HCT116 and SW480 cells were considerably lower than that of control cells using Western blot Figure . Cell prCIP1/WAF1), CDKN1B (p27KIP1), CDKN2A (p16INK4), CDKN2B (p15INK4b), CDKN2C (p18INK4C), CDKN3, CDK4, CDK7 and CDK8. We observed that CDKN1A (p21CIP1/WAF1), CDKN1B (p27KIP1), CDKN2A (p16INK4), CDKN2B (p15INK4b), CDKN2C (p18INK4C) and CDKN3 were significantly elevated when PRMT5 was interfered in both HCT116 and SW480 cells , whereas the mRNA expressions of these genes were not significantly changed in noncancerous NCM460 colon cells (INK4b) was the most significantly upregulated target in PRMT5-deficient HCT116 and SW480 cells compared to that in the control cells. Consistent with this result, protein levels of CDKN2B (p15INK4b) were also found to be remarkably upregulated in PRMT5-depleted HCT116 and SW480 cells by Western blot , further supporting a potential role of PRMT5 in the transcriptional repression of CDKN2B (p15INK4b) expression.Because PRMT5 is a well-established modulator of gene expression, we speculate that one mechanism by which PRMT5 could be modulating CRC progression was through transcriptional control. To investigate this, quantitative real-time PCR analysis was applied to identify downstream transcriptional targets of PRMT5 in HCT116 and SW480 cells. The mRNA expression changes of genes involved in the regulation of cell proliferation and cell cycle were determined, including CDKN1A (p21on cells . Of partP < 0.01). PRMT5 is known to exert its transcriptional regulatory function by symmetric di-methylation of histone H4 at R3 (H4R3me2s) or histone H3 at R8 (H3R8me2s). Given the fact that PRMT5 substantially attenuated the activity of luciferase reporter gene, it would also be of interest to determine whether PRMT5-deposited H4R3me2s and H3R8me2s, which were identified as transcriptionally repressive histone marks P < 0.01). Collectively, our results suggest that CDKN2B (p15INK4b) is a direct transcriptional target of PRMT5 in CRC cells.Of particular interest, CDKN2B promoter-driven luciferase reporter-gene assays showed that the luciferase activity dramatically decreased when HCT116 and SW480 cells were co-transfected with pGL3.0-promoter plasmids containing promoter fragment of CDKN2B (-2000 ~ -1 bp) and pCMV-PRMT5 vectors, compared with that of cells co-transfected with pGL3.0-promoter and pCMV-vector . To further support this, we went on to perform in vivo experiments with mouse xenograft models. Knockdown of CDKN2B (shPRMT5 + shCDKN2B), at least in part, reversed the growth defect in PRMT5-depleted HCT116 cells (shPRMT5) in vivo -tagged PRMT5, which was expressed in the E. coli strain BL21 (DE3) expression system. Strikingly, GST-pulldown assays revealed that GST-tagged PRMT5 (as bait protein) could interact with EZH2 directly, whereas GST alone did not interact with EZH2  experiments, we show that Flag-tagged PRMT5 interacts with endogenous EZH2 in HCT116 and SW480 cells Figure . To estaINK4b) in EZH2-depleted HCT116 and SW480 cells . As expected, both protein and mRNA expression levels of CDKN2B (p15INK4b) were remarkably elevated in EZH2-deficient HCT116 and SW480 cells compared to the control cells, further supporting a potential role of EZH2 in the transcriptional modulation of CDKN2B (p15INK4b) expression. Furthermore, we determined the endogenous expression of EZH2 in NCM460, HCT116 and SW480 cells using Western blot (P < 0.05 & **P < 0.01). The polycomb group protein EZH2, which specifically catalyzes the tri-methylation of lysine 27 of histone H3 (H3K27me3), is well established as a transcriptional repressor P < 0.01). It would also be of interest to determine whether PRMT5 loss affects the depositions of EZH2 and H3K27me3 at the core promoter region of CDKN2B gene. As shown in P < 0.05 & **P < 0.01). Meanwhile, the depositions of PRMT5, H4R3me2s and H3R8me2s at the core promoter region of CDKN2B gene were also significantly reduced when EZH2 was silenced in HCT116 and SW480 cells (P < 0.01). One possible explanation for this phenomenon is that the interaction of PRMT5 and EZH2 may have a crucial role for maintaining their methyltransferase activities. Altogether, these results indicated that EZH2 and its catalytic histone mark-H3K27me3 were deposited at the core promoter region of CDKN2B gene, strongly demonstrating that CDKN2B (p15INK4b) is a direct transcriptional target of EZH2 in CRC cells. Interestingly, we also found that the catalytic marks  of PRMT5/EZH2 were also enriched at the core promoter regions of CDKN1A (p21CIP1/WAF1), CDKN1B (p27KIP1), CDKN2A (p16INK4) and CDKN2C (p18INK4C) genes (P < 0.01). These results demonstrated that a cohort of cell cycle-related genes, including CDKN2B (p15INK4b), CDKN1A (p21CIP1/WAF1), CDKN1B (p27KIP1), CDKN2A (p16INK4) and CDKN2C (p18INK4C) were regulated by PRMT5/EZH2 complexes.We next want to determine if EZH2 was directly regulating CDKN2B expression. Real time PCR analysis was performed to determine the protein and mRNA expression levels of CDKN2B p15INK4b in ern blot A. We fouern blot B. SubseqP < 0.01). Subsequently, in vivo experiments with mouse xenograft models showed that knockdown of CDKN2B significantly reversed the growth defect in EZH2-depleted HCT116 xenografts .To determine whether EZH2 promotes cell proliferation of CRC cells through repressing CDKN2B expression, we knocked down the expression of CDKN2B in EZH2-deficient HCT116 and SW480 cells Figure E. Similas Figure H, I & J,s Figure K. Moreovin vitro studies. Western blot analysis clearly showed that the selected combination of PRMT5i (GSK591) and EZH2i (GSK126) robustly activated the protein expression of CDKN2B .Given that PRMT5/EZH2 complex promotes CRC cell proliferation through epigenetic repression of CDKN2B, we sought to test whether combined inhibition of PRMT5 and EZH2 would exert a better therapeutic effect in CRC cells. To this end, we initially examined the effect of a combination of PRMT5i  and EZH2i  on the proliferation of CRC cells. Heatmap and combination index (CI) plots of combination treatment of PRMT5i (GSK591) with EZH2i (GSK126) showed that the two inhibitors synergistically improved the anti-proliferative effect on HCT116 and SW480 cells . The mRNA expression levels of PRMT5, EZH2 and CDKN2B in HCT116 xenograft tumors treated with PRMT5i, EZH2i or combined treatment with PRMT5i and EZH2i were verified by real-time PCR  in different cohorts of datasets (P = 0.0457), tumor stage (P = 0.0172) and distant metastasis (P = 0.0381). The higher EZH2 expression was correlated with tumor size (P = 0.0337) and tumor stage (P = 0.0187). Survival analysis suggested that the elevated expression levels of PRMT5 and EZH2 were notably associated with worse outcome of patients with CRC through Kaplan-Meier analysis . Nevertheless, low CDKN2B expression was correlated with poor overall survival (P < 0.01), implying a role as tumor suppressor in CRC. In addition, Pearson's correlation analysis was performed to validate the regulatory effect of PRMT5 and EZH2 on CDKN2B expression in CRC samples. As presented in Figure 2 = 0.3411, P < 0.01) and EZH2  were negatively correlated with CDKN2B expression in CRC samples, respectively. Meanwhile, PRMT5 was positively correlated with EZH2 . All these findings suggested that PRMT5 and EZH2 upregulation were partly responsible for the reduced CDKN2B expression in CRC.We retrieved Oncomine database and found PRMT5 (4 upregulated and 0 downregulated datasets) and EZH2 (12 upregulated and 0 downregulated datasets) differentially expressed which are upregulated in CRC tissues  and repression of EIF4E and FGFR3 in CRC cells PRMT5, potentially via H4R3me2s and H3R8me2s deposition at gene promoters, leads to transcriptional repression of pivotal tumor suppressor genes and has been extensively characterized as an oncogene in human malignancies Previous studies established that PRMT5 is associated with ATP-dependent chromatin remodelers , co-repressors , developmental modulators  and transcription factors/co-activators  INK4b) was validated as a direct transcriptional target of PRMT5/EZH2 complex in CRC. CDKN2B/p15INK4b is a member of the INK4 family of cyclin-dependent kinase inhibitors, which has been well established as a bona fide tumor suppressor in a variety of human malignancies INK4b expression in human diseases, especially in cancers. Staller and colleagues have shown that CDKN2B was repressed by Myc via interacting with Miz-1 in primary mouse embryonic fibroblasts CIP1/WAF1), CDKN2C (p18INK4C), CDKN1C (p57KIP2), p63 and PTEN, were identified as PRMT5 targets in gastric cancer by Liu and colleagues, PRMT5 depletion did not appear to affect the expression of CDKN2B, implying that the mechanisms by which PRMT5 regulates cancer cells in CRC and gastric cancer are different Remarkably, another important finding of this study is that cyclin dependent kinase inhibitor 2B (CDKN2B/p15INK4b) axis plays a pivotal role in CRC progression. PRMT5 and EZH2 reciprocally interact with one each other and collaborate as a functional unit, providing a new transcriptional regulation model and a new molecular basis for the interplay between histone lysine methylation (H3K27me3) and arginine methylation (H4R3me2s and H3R8me2s) in epigenetically repressing CDKN2B (p15INK4b) expression (Figure in vitro and in vivo. Altogether, our findings not only reveal important insights that link arginine methylation and lysine methylation to directly modulate CDKN2B transcription, but also pave the way to revolutionize our therapeutic options by blocking either PRMT5/EZH2 activities or the associated transcription regulatory network to treat CRC and even probably other cancers.The findings of our work, thereby, reveal that the PRMT5/EZH2/CDKN2B (p15Supplementary figures and tables.Click here for additional data file.


FLNA, the gene encoding filamin A. MNS is inherited in an X-linked dominant manner. In this study, we describe three members of the same family with MNS, who exhibited different phenotypic severity despite having an identical FLNA gene mutation.Melnick-Needles syndrome (MNS) is an extremely rare osteochondrodysplasia caused by a mutation of FLNA, c.3578 T > C, p.Lys1193Pro, which confirmed the diagnosis of MNS. Her older sister and mother had minimal deformities of the axial and extremity skeleton, but genetic analyses revealed the same FLNA mutation as the patient. The mutation identified in this family has not been previously reported.The patient was 16 months old, with a history of delayed physical development, multiple upper respiratory infections and otitis media episodes. She was referred to our orthopedic clinic because of bowed legs and an abnormal plain chest radiograph. Both upper and lower extremities were bowed. Plain X-rays showed thoracolumbar kyphoscoliosis, with anterior and posterior vertebral scalloping, and thin, wavy ribs. Hypoplasia of the pubis and ischium, with bilateral coxa valga, were also noted. Target exome sequencing revealed a heterozygous mutation of This report illustrates the potential inherited nature of MNS and the phenotypic variability of clinicoradiologic characteristics. In patients with traits suggestive of MNS, a careful medical and family history should be obtained, and genetic testing should be performed for the patient, as well as all family members. FLNA gene (OMIM: #30017) which encodes filamin A. Patients with MNS typically have unusual facial features, short ribbon-like ribs, scoliosis, bowing of the long bones, and vertebral scalloping [Melnick-Needles syndrome  is an extremely rare osteochondrodysplasia –3. To daalloping .MNS is a member of a group of five X-linked diseases with overlapping clinical phenotypes, known collectively as otopalatodigital syndrome (OPS) spectrum disorders . Other mFLNA gene mutation.Because MNS is extremely rare and some cases are lethal, mostly it is detected as de novo condition, but once it is developed, it is inherited in an X-linked dominant manner , 16. HowA 16-month-old female was referred to our orthopedic department because of bowed legs and an abnormal chest X-ray. She was 80 cm (50th percentile) tall and weighed 9.7 kg (25th percentile). Her past medical history was positive for delayed physical development, as well as recurrent respiratory tract infections and episodes of otitis media. Examination revealed several facial characteristics of MNS, including prominent eyes, supraorbital hyperostosis, full cheeks, and micrognathia  and lumbar scoliosis, but she had minimal leg deformities Fig. . Her oldFLNA gene, which encodes the cytoskeletal protein filamin A. FLNA comprises 48 exons and encodes a modular protein with an N-terminal actin-binding domain and a tail of 24 structurally homologous repeats [FLNA gene and has an X-linked pattern of inheritance. They also noted that FLNA mutations are responsible for OPS type 1, OPS type 2, FMD, and TODPD . Interestingly, the mutations identified in this family have not been previously reported in the National Center for Biotechnology Information’s ClinVar. As mentioned earlier, pathogenesis of MNS has not been established well. Further research is also needed to determine pathogenicity of this mutation among these families with MNS.It is not yet known why phenotypes may differ between females with MNS. Skewed X-inactivation and somatic mutation have been suggested as potential mechanisms , 24. ForIt should be noted that the patient with MNS would demonstrate the typical bone deformity in the distal femur, which was called as the Erlenmeyer flask deformity like Camurati-Engelmann disease . CED is another rare genetic skeletal disorder caused by tumor growth factor-β1 mutation, which is characterized by limb pain, muscle emaciation and weakness, cortical thickening of the diaphysis of long bones, and also Erlenmeyer flask deformity , 26.Although MNS is rare, physicians should be aware of the disorder, including its variable manifestations, because of the potential lethality of severe disease. A possible hereditary mutation should be suspected even when family members have an almost normal appearance. Thus, in patients exhibiting traits suggestive of MNS, imaging studies and genetic testing should be performed for both the patient and all family members.Informed written consent was obtained. It contains publication of this report and the accompanying images, including photographic rights of patient, her mother and her sister (Use of medical information for academic purposes, including the portrait rights shown in Figs.


The study facilitates digital nomadism for tourism research and recognizes a unique product offer on the market: the combined coworking and coliving space in compelling or exotic destinations. The aim of the study is to explore the experience of coworking and coliving by digital nomads and identify valuable elements. Qualitative interview data are used to analyse combined coworking and coliving space environments from the perspective of digital nomad tourists. A better understanding of digital nomad preferences may help destinations and business owners to attract digital nomads during and after the pandemic. The study’s findings, perceived advantages and disadvantages of coworking and coliving spaces, may serve as a guideline for targeting digital nomads. Numerous coworking hotspots worldwide attract digital nomads; some examples include Bali, Lisbon, Mexico City, Barcelona. A more recent trend is in work-travel coliving spaces as a part of the coworking in exotic environments or places promising exploration of a new place or benefits of warm weather, beaches, or a calm environment for recreation . This trThe destinations and hospitality industry may benefit from gaining a better understanding of the role of coworking and coliving for digital nomad tourism. It is worth opening a conversation about the new type of attractions to facilitate digital nomad travel’s institutionalization for tourism research. This study aims to explore the experience of a combined coworking and coliving space during travel and acknowledge elements that digital nomads most value. The study questions which factors affect digital nomad’s choice to stay in the coworking and coliving space.Coworking and coliving space belong to the sharing economy, where the value is co-created , 5. The Establishing of coliving spaces was a market response to the labor conditions crisis that later influenced the conventional meaning of home, making it a co-productive and co-emergent practice: instead of being an escape from work, it is productive; instead of being private, it is social; and instead of being inhabited long term, it is mobile . The proNevertheless, for digital nomads in travel destinations, coliving space as part of a coworking space commonly has a one-week minimum stay requirement, making it a less flexible option than room renting or a hotel. The majority of the coliving spaces in inspiring destinations target digital nomads in their marketing activities, and it reflects the new meaning of a home through multiple short-term tenancies across the globe, home-as-work, and home as a social network . One of This study investigates the experience of staying in coworking and coliving space during travels following a qualitative research design by using inductive content analysis. Semistructured interviews were completed online in May 2020 to collect data for the study. Participants were asked to reflect on their experience in coworking and coliving space, share what they would have improved and future travel preferences. The participants’ search was performed via social media accounts of different coworking and coliving companies and targeted travelers who experienced them abroad. The principle of saturation was applied to distinguish the sample size. In total, 12 participants were involved in the study, representing five nationalities, covering coworking and coliving experience in Asia, Europe, Africa, South, Central and North America. Data collection and data analysis were conducted simultaneously. The data was analyzed by one researcher, which is common for inductive content analysis .“There is sort of that social pressure, people want to talk to you. There is this sort of expectation that people are friendly and want to network. [. . .] I guess I would make it a little bit more private.”This section summarizes ten main themes drawn in regards to coworking and coliving experience. The most recognized advantage is a community sense that includes easy access to meeting people and events; this finding is aligned with previous research –10. In cThe main perceived advantages and disadvantages of coworking and coliving spaces were noted and summarized in Table Interestingly, the advantage of convenience comes with a price; segregation from local people and culture. Four participants mentioned distancing from a local community, with one person comparing coworking and coliving space with a Disneyland experience. Digital nomads being separated from locals was also found in previous studies , 18.Another recognized area is the pleasant work environment of the coworking space. Participants pointed out such features as comfortable furniture, good lighting, plants, air-conditioning, light, noise level, internet speed, and coffee quality. The importance of the atmosphere and interior aesthetics and the layout of the space was previously mentioned by researchers . BesidesThe following theme emerged is that coworking space gives business advice and is a place to learn from others; this advantage was mentioned by Capdevila  when he An additional key issue recognized is related to cost; the price of the coliving option being higher than other accommodation types of the same quality. As previous literature revealed, for a coliving concept, community, flexibility and price are among attractions . However“Coliving I stayed in had a slightly higher price point, which I think helped filter out (people) [. . .] I think the higher price point sort of filtered out some of the people who were more on like “I don’t really know what I’m doing, sort of backpacker type of vibe.”However, some participants saw a positive influence of a higher price:“I’ve only been in coliving space once but only to try it I didn’t like it because it was so expensive.”Others perceived the facilities being overpriced. One shared:“I was there for about a month and a half when they started doing coliving and I went to view the houses (coliving). It was more expensive to stay in a coliving space then to stay in a hotel. And that was ultimately the factor.”A different participant explained his/her reasons to moving to a hotel:The next thread recognized is that coworking and coliving spaces provide helpful local advice, especially when first time in the destination. This was an exciting finding, as, besides a vast online community within remote workers who travel, many participants shared that they do not plan much in advance and refer to coworking and coliving spaces to find places to eat and things to do. From the tourism perspective, this may be a new opportunity for local businesses to recognize coworking and coliving spaces as information intermediates for travelers and establish partnerships within locals.One more advantage is in the pulling ability of coworking spaces to attract digital nomads. Out of all participants, three mentioned that they are looking specifically for a combined coworking and coliving space in the destination, even choosing a destination based on it. It is an exciting new finding for destination managers and a new opportunity for emerging destinations. Surprisingly, these three participants were the most experienced travelers.“I did not belong in a predominantly white community. So that’s why I chose a community or a coworking space based on the community as I test it out. I tried out several coworking spaces, but I tested it out based on “how do I do as an Asian?”Another matter that emerged is in different perceptions of the digital nomad community by travelers from Asia. As most digital nomad travelers are Western, Asian nomads, when traveling, had a different community and travel experiences. Asian travelers in sport-induced tourism previously observed similar differences compared to their Western counterparts . Partici“Most of the places I was perhaps the only Indian who was traveling while working a lot of people told me that I was the only Indian, so having some friends from home along to accompany with me was not an option because I was the only one I knew who could do this.”Another participant said:The last theme is related to the importance of the first impression, so-called vibe, or energy of the place that comes mostly from people. Five participants recognized that they draw conclusions about the space from the first few moments in a place, and participants even compared it with the first day in school or mentioned that it is like love at first sight. This first impression was often studied in hospitality and was found to be valuable , 23. HowThis paper contributed to the body of knowledge by identifying elements that are valued in coworking and coliving space during travel. The content analysis of semistructured interviews with digital nomads who stayed in coworking and coliving space assisted in disclosing their travel experience. As a result, a summary of the advantages and disadvantages of coworking and coliving space seen by digital nomads was developed Table . This stAmong the practical implications of this study, perhaps the most important one is that coworking and coliving space can be an attraction of destinations and serve as a peculiar tourism information center. It suggests to the tourism boards who focus on digital nomads to invest in relationships with coworking businesses, just as it is commonly practiced with theme parks and hotels. The findings may serve as a guideline for destinations and business owners to develop new products, adjust marketing strategy, and improve the service quality.The limitation is related to the range of coliving spaces covered in the study. As community-based coliving may have different forms, there are unique retreat farms, converted Buddhist temples, community-based eco-villages, and many others. However, the study has initially reached out to travelers who stayed in establishments that call themselves a coliving space (as part of the coworking space) and focused the study around them. Future research is worth developing a categorization of coworking and coliving spaces for travelers and factors influencing a particular establishment’s choice during travels, such as length of stay and trip composition.Out of the current study’s scope, all participants indicated that they were looking forward to their next adventure once travel restrictions are down; some of them even were still travelling during data collection (in May 2020) and stayed in the coworking and coliving space at the moment of the interview. Digital nomad tourism can continue to be the world’s significant phenomenon after the mobility change of COVID-19. More future research on understanding digital nomads as a segment for tourism is called for.


Aplysia californica, we investigated the molecular processes governing the interactions between sensory neurons and both target and non-target motor neurons during synapse formation in culture. We found that evolutionarily-conserved intercellular and intracellular signaling mechanisms critical for learning-related plasticity are also engaged during synaptogenesis in this in vitro model system. Our results reveal a surprising bidirectional regulation of molecular signaling between sensory neurons and non-target motor neurons. This regulation is mediated by signaling via both paracrine and autocrine diffusible factors that induce differential effects on transcription and on protein expression/activation in sensory neurons and in target and non-target motor neurons. Collectively, our data reveal novel molecular mechanisms that could underlie the repression of inappropriate synapse formation, and suggest mechanistic similarities between developmental and learning-related plasticity.The formation of appropriate neural connections during development is critical for the proper wiring and functioning of the brain. Although considerable research suggests that the specificity of synapse formation is supported by complex intercellular signaling between potential presynaptic and postsynaptic partners, the extracellular factors and the intracellular signal transduction pathways engaged in this process remain largely unknown. Using the sensory-motor neural circuit that contributes to learning in defensive withdrawal reflexes in  The molecules mediating these intercellular exchanges, including growth factors, neuropeptides, neurotransmitters, and cell adhesion molecules, activate intracellular signaling cascades leading to functional and structural changes that promote the formation of functional synapses5. The synaptic proteins supporting the morphological changes that mediate the formation, restructuring, and elimination of synapses have been extensively studied5. However, the extracellular factors and the intracellular signal transduction pathways that regulate the expression of the genes encoding proteins required for appropriate synaptic connectivity remain largely unknown.The formation of specific neural connections is critical for the proper development and function of the nervous system. Specificity of synapse formation is supported by complex bidirectional intercellular and intracellular signaling between and within potential pre- and postsynaptic partners6. Indeed, in the last two decades, research examining developmental and learning-related plasticity has uncovered a striking degree of overlap between the molecular mechanisms employed by the brain during these different biological processes10. For example, the members of the neurotrophin family of growth factors were initially characterized as critical regulators of neuronal survival, differentiation, and growth12, but were later shown to contribute to the molecular events underlying activity-dependent synaptic growth and restructuring in the mature nervous system13. These findings raise important questions about the mechanistic similarities between neural development and adult plasticity, and offer opportunities for integrating findings from both fields of research.Over a century ago Santiago Ramón y Cajal proposed that the processes underlying the development of the nervous system and the neural plasticity supporting learning and memory in adulthood share fundamental mechanismsAplysia californica is a useful model system for studying the cellular and molecular mechanisms underlying these forms of plasticity14. In Aplysia, facilitation or depression of monosynaptic connections between identified presynaptic sensory neurons (SNs) and postsynaptic motor neurons (MNs) contributes significantly to different forms of learning that can be induced in simple defensive withdrawal reflexes16. Importantly, this SN-MN microcircuit demonstrates synapse-specificity and can be reconstituted in culture17. In vitro, as in vivo, Aplysia SNs selectively form chemical synapses with their physiological target MNs (L7 MNs), but do not form synapses either with themselves or with non-target MNs (L11 MNs)  growth factor and neuropeptidergic signaling, (ii) protein kinase activation, and (iii) cyclic-AMP response element binding protein (CREB)-dependent transcription. Our results reveal a surprising bidirectional regulation of molecular signaling between Aplysia and other systems26. To explore transcriptional effects of SNs on MNs, we cultured non-target L11 MNs and target L7 MNs in isolation or in contact with SNs . However, there was no significant difference in C/EBP mRNA levels between isolated and paired target L7 MNs . These results suggest that SNs downregulate C/EBP mRNA levels in non-target MNs, but not in target MNs.We first investigated whether SNs regulate transcriptional events in both non-target and target MNs Fig. . We focuSNs Fig. . SNs fasC/EBP expression in MNs? To address this question, we cultured non-target L11 MNs either in isolation or in the same culture dish with SNs (conditioned MNs), without physical contact between MNs and SNs . These data show that the mRNA regulation exerted by SNs on non-target MNs is not mediated by contact-dependent interaction between neurons, but, rather, is induced through the actions of diffusible factor(s) secreted by SNs.We next explored the molecular mechanisms mediating the observed mRNA regulation between mismatched synaptic partners. We first asked the question: Is physical contact between SNs and non-target MNs necessary for the downregulation of SNs Fig. . We foun4. Signaling through the tropomyosin receptor kinase B (TrkB), in particular, has been shown to act through the cyclic-AMP pathway to regulate neural development in mammals28, and long-term synaptic plasticity and memory in both mammals30 and Aplysia32. The requirement of TrkB signaling for the molecular, cellular, and behavioral expression of long-term memory in Aplysia has been shown pharmacologically by blocking endogenous TrkB signaling with soluble TrkB receptor bodies, TrkB-IgG, which sequester secreted BDNF/NT4-like ligands and prevent them from binding to endogenous TrkB-like receptors and inducing intracellular signaling32. Thus, we used the TrkB-IgG receptor bodies in our experiments to test the hypothesis that a TrkB ligand is mediating the mRNA regulation effects of SNs on non-target MNs. Towards this end we cultured isolated or SN-conditioned non-target and target MNs in the presence of either control- or TrkB-IgG  = 11.25, p = 0.001; one-tailed Dunnett’s multiple comparisons test: Isolated L11 MNs: 1 + 0.11, n = 11; Conditioned L11 MNs + IgG: 0.67 + 0.06, n = 8; p = 0.04, t15 = 2.422). To our surprise, C/EBP mRNA levels in TrkB-IgG-treated conditioned non-target L11 MNs were significantly higher than those in isolated L11 MNs . This pattern of results was specific to non-target L11 MNs, as a one-way ANOVA analysis did not show a significant difference between group means in the target L7 MNs condition  = 2.28, p = 0.321; Isolated L7 MNs: 1 + 0.3, n = 8; Conditioned L7 MNs + IgG: 1.33 + 0.26, n = 8; Conditioned L7 MNs + TrkB-IgG: 1.42 + 0.22, n = 7). Importantly, the effects of TrkB-IgG treatment were specific to conditioned non-target L11 MNs, and were not observed in isolated non-target L11 MNs . Collectively, these results reveal three features of the engagement of TrkB signaling in the SNs-induced regulation of C/EBP expression in MNs: (i) TrkB signaling is required for SNs-induced C/EBP downregulation in non-target MNs; (ii) in the absence of TrkB signaling, SNs upregulate C/EBP expression in non-target MNs, but not in target MNs; and (iii) TrkB signaling-dependent regulation of C/EBP mRNA levels in non-target MNs is contingent on signaling from SNs.Growth factors are extracellularly released proteins that mediate both developmental and learning-related plasticityC/EBP downregulation in non-target MNs, but, to our surprise, also revealed an independent diffusible signaling pathway from SNs that facilitates C/EBP expression in the absence of TrkB signaling. Sensorin is a SN-specific secreted neuropeptide in Aplysia that has been shown to play critical roles in both synapse formation between SNs and target L7 MNs24, and in memory-related synaptic plasticity35. Thus, we tested the hypothesis that sensorin signaling mediates the SN-induced C/EBP regulation in non-target MNs. In these experiments, isolated non-target L11 MNs or target L7 MNs were cultured in the presence of (i) vehicle, (ii) sensorin peptide, and (iii) control- or TrkB-IgG. Treatment with sensorin peptide did not have a significant effect on C/EBP expression in isolated non-target L11 MNs  = 12.9, p = 0.001; two-tailed Dunnet’s multiple comparisons test: L11 MNs + vehicle: 1 + 0.13, n = 20; L11 MNs + sensorin + IgG: 0.79 + 0.07, n = 18; p = 0.456, t28 = 1.349). However, in the presence of TrkB-IgG, treatment with sensorin peptide significantly upregulated C/EBP expression in isolated non-target L11 MNs . Moreover, there was a significant difference in the effects of sensorin on C/EBP expression between control IgG- and TrkB-IgG-treated L11 MNs . As predicted from the lack of SN regulation of C/EBP expression in target L7 MNs, one-way ANOVA analysis did not show significant differences between the vehicle, sensorin, and control or TrkB-IgG-treated groups in the target L7 MNs condition  = 2.872, p = 0.07; L7 MNs + vehicle: 1 + 0.1, n = 17; L7 MNs + sensorin + IgG: 1.17 + 0.11, n = 16; L7 MNs + sensorin + TrkB-IgG: 0.81 + 0.09, n = 14). These findings indicate that sensorin signaling alone is not sufficient to significantly downregulate C/EBP mRNA levels in the presence of TrkB signaling in non-target MNs . Interestingly, sensorin signaling is sufficient to induce C/EBP upregulation in the absence of TrkB signaling in non-target MNs, an effect comparable to that observed in non-target MNs cultured in the presence of SNs  whether SNs affected MAPK activation in non-target and target MNs and (ii) if so, whether TrkB signaling was engaged. Our results showed a significant increase in MAPK activation in conditioned non-target L11 MNs when compared with isolated L11 MNs  = 4.037, p = 0.029; two-tailed Tukey’s multiple comparisons test: Isolated L11 MNs: 100 + 15.7%, n = 10; Conditioned L11 MNs: 147 + 8.5%, n = 10; p = 0.024, t26 = 3.981). While modestly reduced, this effect was not significantly disrupted by treatment with TrkB-IgG, suggesting that TrkB signaling does not play a major role in the SN-induced MAPK activation in non-target MNs . Furthermore, there was no significant difference in MAPK activation between isolated and conditioned target L7 MNs . Collectively, these results show that SNs induce an increase in MAPK activation in non-target MNs, but not in target MNs.We next investigated a signal transduction mechanism that could be upstream of the mRNA regulation events we observed thus far. The activation of mitogen-activated protein kinase (MAPK), a well-characterized second messenger cascade that links extracellular signaling to transcriptional events in the nucleust-test: L11 MNs + vehicle: 100 + 13.8%, n = 15; L11 MNs + sensorin: 140 + 11.1%, n = 12; p = 0.038, t25 = 2.184), but not in target L7 MNs . Taken together, these results indicate that sensorin secreted by SNs induces an increase in MAPK activation specifically in non-target MNs.We next examined whether sensorin signaling mediates the SN-induced MAPK activation in non-target MNs. To address this question, we cultured isolated non-target and target MNs in the presence of vehicle or sensorin peptide. Treatment with sensorin peptide led to a significant increase in MAPK activation in non-target L11 MNs  = 12.34, p < 0.0001; two-tailed Tukey’s multiple comparisons test: Isolated SNs: 100 + 3.5%, n = 22; SNs + L11 MNs: 131 + 3.7%, n = 20; p < 0.0001, t75 = 7.68), and this increase was not blocked by treatment with TrkB-IgG . Interestingly, sensorin protein levels were also significantly higher in SNs cultured in the presence of target L7 MNs when compared with isolated SNs . These data suggest that both non-target and target MNs upregulate sensorin protein levels in SNs.Our data thus far show that SNs induce both mRNA downregulation and protein kinase activation specifically in non-target MNs. In a final set of experiments, we investigated the converse, examining whether non-target and target MNs induce molecular changes in SNs. Given our previous findings, we examined changes in sensorin protein levels using immunofluorescence in SNs cultured in isolation, or in the presence of non-target or target MNs. We found that cytoplasmic sensorin protein levels were significantly increased in SNs cultured in the presence of non-target L11 MNs when compared with isolated SNs  paracrine neuropeptidergic signaling from SNs  SNs secrete the neuropeptide sensorin, which binds to receptors on non-target MNs and induces an increase in both MAPK activation and C/EBP expression . (2) In response to sensorin signaling , non-target MNs secrete a TrkB ligand which acts in an autocrine manner to downregulate C/EBP expression . (3) An unidentified secreted factor from non-target MNs binds to receptors on SNs and induces an increase in sensorin protein levels; this MN-induced increase in sensorin levels can, in turn, initiate and/or prolong the sensorin secretion in Step (1), creating a positive feedback loop for sensorin signaling. This model of intercellular and intracellular molecular regulation between mismatched synaptic partners reveals novel and evolutionarily-conserved molecular mechanisms that could underlie the specificity of synapse formation in this Aplysia neural circuit, as well as in other neural systems.In the present study we investigated the molecular interactions between SNs and their appropriate and inappropriate target MNs. Our results reveal an unexpected and complex bidirectional regulation of intracellular signaling between Ns Figs. ,5 and  and receptors mediating these effects, they do show that they are independent of TrkB signaling, at least for the non-target MN effects on SNs. Potential candidates for the observed retrograde signal(s) that increase(s) sensorin protein levels in SNs are the netrin family of chemotropic factors. Interestingly, netrin-1 was shown to promote translation of sensorin mRNA in the neurites of SNs contacting both target and non-target MNs in Aplysia24, and to induce synaptogenesis in C. elegans40. Taken together with previous studies in Aplysia24, our results strengthen the existing evidence for retrograde diffusible signaling from both target and non-target MNs, and describe similar molecular changes induced by the two types of MNs in SNs. It would be interesting for future studies to investigate whether the diffusible signaling molecules from L11 and L7 MNs are the same and whether their actions depend on signaling from SNs.Previous studies in Aplysia co-culture system has reported changes in the cellular distribution of sensorin mRNA in SNs paired with MNs24. For example, an important series of studies showed that both target L7 and non-target L11 MNs induced increases in the expression of sensorin mRNA in the distal neurites of SNs above levels in isolated SNs, although the L7 MN effect was significantly greater than that of the L11 MN22. Furthermore, downregulation of sensorin mRNA in SNs has been shown to strongly inhibit synapse formation between SNs and target MNs22, which requires local translation of sensorin mRNAs24. In addition, L7 MN-dependent release of sensorin from SNs was found to be required for the regulation of sensorin mRNA distribution as well as for synapse formation21. Interestingly, the differences in sensorin mRNA localization between target and non-target MNs were observed after 4 days in culture, but were not detected at earlier time points22. Moreover, sensorin secretion was reported to decline from day 1 to day 4 in culture21. These reports suggest that, although sensorin is secreted from SNs during the early stages of synapse development in vitro, the differences in intracellular events between L7 and L11 MNs, such as sensorin mRNA distribution, are not present until the later stages of synapse maintenance, highlighting changes in the temporal dynamics of sensorin signaling. While synapse formation between matched SNs and MNs occurs during the first hours in culture22, the prevention of synapse formation might engage a prolonged, active process of molecular inhibition between mismatched synaptic partners. In the present study we found that, after 5 days in culture, both target and non-target MNs induce significant increases in somatic sensorin protein levels in co-cultured SNs. Thus, our current findings complement and extend previous studies by showing that diffusible factor(s) secreted from MNs can act over long spatial and temporal ranges to regulate sensorin expression in SNs, even in the absence of physical contact between neurons. It would be interesting for future studies to investigate whether the increase in somatic sensorin protein expression we observed is due to de novo transcription and/or translation, or alternatively, is due to decreased secretion or transport to the neurites.Previous research examining the processes underlying the specificity of synaptogenesis in the Aplysia23 investigated the differences in synaptic protein expression between L7 and L11 MNs and found that both types of MNs expressed comparable basal levels of AMPA- and NMDA-like receptors and of the Dscam protocadherins family of cell adhesion molecules (CAMs). Moreover, culturing SNs with target L7 MNs induced Dscam-dependent clustering of both AMPA and NMDA receptors on the cell surface of L7 MNs. Interestingly, only clustering of NMDA, but not of AMPA receptors, was observed in L11 MNs that were co-cultured with SNs23. CAMs are critical for the trans-synaptic signaling underlying synaptogenesis, and their transcription is tightly regulated during this process5. In addition, postsynaptic CAMs, such as latrophilins and cadherins, have been reported to mediate synapse specificity43. In Aplysia, the postsynaptic protocadherins Dscam were found to be required for de novo synaptogenesis between SNs and L7 MNs and for AMPAR clustering in L7 MNs23. Collectively, these findings suggest the intriguing possibility that L11 MNs express the receptors necessary for glutamatergic synapse formation with SNs, and that an active downregulation process acts to prevent AMPAR clustering in these inappropriate MNs. Differential transcription regulation of CAMs in target and non-target MNs could lead to differences in AMPAR clustering and thus could underlie synapse specificity. Our current findings that signaling from SNs downregulates CREB-dependent transcription in non-target MNs could provide a possible transcriptional mechanism through which CAM-dependent AMPAR clustering is inhibited in non-target MNs.Whether SNs have any effects on MNs, on the other hand, is a less studied question. A study in 1. In the present study we investigated the functional significance of reversing the SN-induced C/EBP downregulation in non-target MNs. We tested electrophysiologically the presence of functional chemical synapses between SNs and target and non-target MNs treated with control- or TrkB-IgG  long-range diffusible signaling through the paracrine actions of sensorin neuropeptide and of unidentified factor(s) secreted by MNs, and (ii) local signaling through the autocrine actions of a TrkB ligand in non-target MNs, could induce permissive and non-permissive states of transcription in target and non-target MNs, respectively, thereby providing target recognition signals that could underlie the specificity of synapse formation.Our results show that, in the absence of TrkB signaling, exogenous application of sensorin peptide to isolated non-target MNs, recapitulates the facilitatory MAPK activation and C/EBP mRNA levels do not address whether the observed regulation represents de novo gene expression or posttranscriptional regulation of mRNA stability49, both of which have been shown to be downstream of MAPK activation50. However, since C/EBP is a transcription factor, the regulation of its mRNA will most likely result in altered transcription of downstream late-response genes that, in turn, could alter the synaptic architecture of target and non-target MNs.Importantly, our data describing the regulation of Aplysia mismatched synaptic partners. Our findings provide evidence highlighting the differences and similarities between the processes mediating the promotion of appropriate neural connections and the prevention of inappropriate ones. Future work investigating earlier time points in this culture system are likely to reveal facilitatory effects of SNs on target MNs that might be mediated by similar signaling mechanisms with the ones we describe here. While little is known about the processes involved in the initial formation of synapses between SNs and MNs during development in this system, our study revealing mechanistic aspects of specificity of synapse formation between cultured adult neurons can inform future developmental research. Furthermore, the evolutionarily-conserved molecular mechanisms we investigated in the context of synaptogenesis, such as TrkB and sensorin signaling, MAPK activation, and CREB-dependent transcription, are known to be engaged during learning-related synaptic plasticity in the same and other systems39. While clear similarities between the two programs of plasticity have been previously described10 and were suggested by our present findings , our results also revealed differences in the valence of transcriptional effects . Taken together with previous research in Aplysia, the present study reveals novel molecular mechanisms of plasticity that could represent points of contact between developmental synapse formation and experience-dependent synaptic restructuring.In conclusion, the results in the present study reveal novel intercellular and intracellular molecular mechanisms underlying the interactions between cultured Aplysia californica from the National Resource for Aplysia Facility at University of Miami were housed in an aquarium for at least 3 days before experimentation. Pleural SNs and abdominal L11 and L7 MNs were identified based on morphological characteristics and location. SNs and MNs were cultured according to a published protocol17 and kept in culture for 5 days. SN-MN pairs consisted of a single MN paired with a single SN (3–4 pairs per plate). Plates with isolated or conditioned neurons consisted of 3–5 MNs cultured in isolation or in the presence of ~30 SNs (conditioned neurons were cultured with ~3 mm distance between SNs and MNs).Adult (70–90 g) and juvenile (1–4 g) TrkB-IgG or control IgG  was added to cultured MNs 20 min before the addition of SNs or sensorin to the culture plates, and was left in the culture medium until RNA extraction on day 5. Sensorin peptide  or Vehicle (0.1% BSA in PBS) was added to isolated MNs on the day of culture and was left in the culture medium until RNA extraction on day 5.Total RNA from single MNs was isolated and purified using the RNAqueous-Micro Total RNA Isolation Kit (Invitrogen). cDNA was synthesized using the SuperScript IV Reverse Transcriptase Kit (Invitrogen). Quantitative PCR was performed using a Roche LightCycler 480 and SybrGreen I Master . The following primers were used: ApGAPDH , ApC/EBP . The amount of each gene was normalized to that of ApGAPDH within the same sample using the ΔΔCt method.Cultured neurons were fixed for 30 min in a solution of 4% formaldehyde in TBS containing 30% sucrose. After 5 washes with TBS, fixed cells were blocked for 30 min at room temperature in a TBS blocking solution containing 10% normal goat serum and 0.2% Triton X-100. Cells were then incubated for 3 nights at 4 °C with anti-sensorin  or anti-phosphorylated MAPK antibody . After 3 washes with TBS, a secondary antibody conjugated to Cy-5  was applied for 2 hrs at room temperature. Cells were then washed 4 times in TBS and mounted using ProLong Gold Antifade Mountant (Invitrogen). Images were obtained with a Leica SP8 confocal microscope using a 63 X oil-immersion lens. Images taken through the middle of the nucleus were used for ImageJ analysis of mean fluorescence intensity in the cell body.Electrophysiological recordings were performed in current-clamp mode on SN-MN co-cultures. Extracellular stimulation of SNs was performed using blunt glass microelectrodes filled with ASW. Intracellular recordings from MNs were performed using 5–12 MΩ glass microelectrodes filled with 3 M KCl. MNs were held at approximately −80 mV by passing constant hyperpolarizing current. SNs were stimulated to elicit a single action potential, and excitatory postsynaptic potentials (EPSPs) were recorded in the follower MNs.t-tests were used. For comparisons of three or more groups, one-way ANOVA followed by planned multiple comparisons tests were used. qPCR data was analyzed using Dunnett’s multiple comparisons tests due to unequal variance between groups. Immunofluorescence data was analyzed using Tukey’s multiple comparisons test due to equal variance between groups. All p values are reported for two-tailed analysis, unless otherwise specified. Statistical significance is defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.All analyses were conducted using GraphPad Prism. All data are expressed as mean + SEM. Parametric statistics were used throughout due to a normal distribution of the data. For two-group comparisons unpaired Student The experiments in this study do not involve human subjects or samples from humans, other vertebrates or higher invertebrates.Supplementary Figure S1