Background/aim:IFNG-AS1 is a long noncoding RNA that works as an enhancer for the Interferon-gamma (IFN-γ) transcript. GAS5 (growth arrest-specific 5) is a lncRNA that is associated with glucocorticoid resistance. Aberrant expressions of IFNG-AS1 and GAS5 are directly linked to numerous autoimmune disorders but their levels in childhood ITP are still obscure. This study aims to elucidate expressions of target lncRNAs in childhood ITP and their association with pathophysiology and clinical features of the disease as well as their association with types and treatment responses.Method: The fold changes of target lncRNAs in blood samples from children with ITP and healthy controls were analyzed using quantitative real-time PCR (qRT-PCR).Results: There were overexpressed lncRNAs IFNG-AS1 and GAS5 in serum of childhood ITP patients  or chronic ITP (>12 months) [ median (IQR) = 5.6 (0.25–12.59) and 5.61 (1.15–16.91) respectively] when compared to newly diagnosed <3 months patients . Also, significant higher lncRNAs IFNG-AS1 and GAS5 were present in patients with partial response to treatment  or non-response [IFNG-AS1 median (IQR) = 4.19 (1.25–22.39) and GAS5 median (IQR) = 5.11 (2.34–15.27)] when compared to patients who completely responded to treatment (IFNG-AS1 median (IQR) = 2.09 (0.2–14.58) and GAS5 (median (IQR) = 2.51 (0.09–10.33). In addition, following therapy, the expressions of IFNG-AS1 and GAS5 are significantly negatively correlated with platelet count.Conclusion: Findings suggest that lncRNAs IFNG-AS1 and GAS5 are novel diagnostic and prognostic genetic markers for childhood ITP that can aid in a precise prediction of the disease’s progress at the time of diagnosis and could be a useful tool for treatment planning. A vast number of non-coding transcripts were discovered during human genome sequencing, they were formerly believed to be non-functional, however recently, scientists discovered that they have regulatory functions on gene expression involving transcriptional, posttranscriptional, and translational levels. Long noncoding RNAs (lncRNAs) are one type of them which defines as RNA transcripts with more than 200 nucleotides that are not translated into protein . As moreIFNG-AS1 (Ifng antisense RNA 1), also known as Tmevpg1 , or NeST, , is affiliated as lncRNA transcript which gene is found on the DNA strand opposite the interferon-gamma (IFN- γ) coding gene so hence its name. The coexpression of lncRNA, IFNG-AS1 with T-bet is related to the active transcription of IFN- γ in Th1 cells. Th1 cells are an inflammatory fraction of CD4+ T cells that primarily generate interferon-gamma (IFN-γ), an inflammatory cytokine that protects against intracellular pathogens, and is related to delayed-type hypersensitivity, and autoimmune diseases including ITP  is a lncRNA that was first discovered as a cancer tumor suppressor gene. GAS5 expression has been found to be abnormal in numerous malignancies, and this gene has been linked to cell cycle inhibition and apoptosis . GAS5 interacts with the DNA binding domain of glucocorticoid receptors (GRs), and preventing GRs from connecting with DNA, preventing glucocorticoid activity which is a powerful immunosuppressive and hence contributes to the development of autoimmune disorders  is a blood disorder characterized by a decreased platelet count below 100×10gulation , (Ayoub gulation .IFNG-AS1 and GAS5 expressions are linked to numerous autoimmune disorders suggesting the possible role of these genes in the pathogenesis of autoimmunity state (Childhood Immune Thrombocytopenia (ITP) is one of the most frequent autoimmune hemorrhagic diseases affecting 5 to 10 children out of every 100,000. Twenty to thirty percent of cases were shown to be prone to chronicity with increased susceptibility to bleeding, infection, and blood malignancy than healthy children  Li et a. Previouty state .IFNG-AS1and GAS5 in peripheral blood of adult ITP patients  and GAS5  and we used GAPDH primer  to standardize the expression pattern and quantify the target long non-coding RNAs.Materials used to execute this work include; one- MiRNeasy Serum/Plasma extraction kit  was used for total RNA extraction from serum. Two- The RT2 First Strand Kit  was used for reverse transcription to produce cDNA. Three- The RT2 SYBR Green PCR kit  was used to perform qRT-PCR along with specific primers supplied by Qiagen, ; for This case-control study was comprised of eighty-eight children (1–13 years old) who suffered from primary ITP and eighty-eight healthy age and sex-matched children with no history of any disease. Cases were gathered from the Pediatric Departments’ inpatient and outpatient clinics at Fayoum University Hospital in Egypt over a period of 6 months between Jan 2022 and June 2022.helicobacter pylori infection history, HCV and/or HBV viral infection. Causes of secondary ITP such as autoimmune rheumatological diseases and the previous treatment regimen. A systematic clinical examination is performed, with special attention paid to clinical symptoms of bleeding such as bruises or purplish spots on the skin, mucous membranes, or gums, and any concomitant splenomegaly. Laboratory tests including complete blood count (CBC) and bone marrow examination to exclude other diseases resulting in thrombocytopenia were done. The definite diagnosis was established as primary idiopathic thrombocytopenia depending on the presence of solitary low platelet count (<100×109/L) in a patient with multiple bruises and/or petechiae.A detailed history was taken including age, sex, complaint at presentation, disease duration, presence of bleeding manifestations, history of splenectomy, presence of preceding febrile illness, recent vaccination, 9/L without clinically significant bleeding, 43 (48.87%) showed partial response to treatment (PR); which is a platelet count between 30 and 100×109/L or twice the baseline platelet count without significant bleeding, and 7 (7.95%) with no response (NR) in patients with the present spleen is platelet count <30×109/L in two different measurements or the increase in platelet is less than twice baseline count  were newly diagnosed <3 months, 27 (30.86%) with persistent ITP 3–12 months, and 19 (21.60%) had chronic >12 months ITP. Treatment options regimen includes corticosteroids, IVIG, immunosuppressive drugs, and Eltrombopag . This work was done per the Declaration of Helsinki’s ethical requirements.With the help of skilled medical staff, we withdrew 5 mL of venous blood from each participant into a plain tube for serum preparation and RNA extraction. For serum separation, samples were left on a straight surface for nearly 15 min, supernatants were transferred to clean tubes and centrifuged at 4,000 xg for 10 min to isolate serum which was kept at -80°C until use.g for 15 s, discarded the flow-through and transferred the spin column containing binding RNA to clean 2 ml collection tubes. We used the washing buffers RWT and RPF in the kit; each spin column received 700 μl of RWT buffer, then centrifuged at 8,000 xg for . The first step is to clean the area of work, pipettes, and centrifuge with prepared ethanol 70%, second step is aimed at lysis of cells and release of nucleic acid so after wearing clean gloves we added 1,000 μl QIAzol lysis reagent to 200 μL sample and incubated the mix after shaking vigorously at room temperature for 5 min. Thirdly, to enhance phase separation so that RNA is purified from DNA and protein debris, we added 200 μl chloroform, vortexed, and put the samples in the freezer for 5 min to avoid RNA destruction then the samples were centrifuged at 12.000 xg for 15 min. Then, we collected the upper aqueous parts in new tubes and added 1.5% of its volume to 100% ethanol to promote RNA precipitation. Afterward, we used the mini spin column in a 2 ml collection tube accompanied RNeasy kits to promote solid phase separation of RNA by adding the samples into two subsequent sessions (750 μl) each with centrifugation at 8,000 0 xg for  s, the fThe RT2 First Strand Kit  was used for reverse transcription to produce cDNA, and the manufacturer’s procedure was followed. RT reaction was done in a final volume of 20 μl (10 μl reverse-transcription mix was added to each tube containing genomic DNA elimination mix), Conventional PCR was used to incubate the samples for 60 min at 37°C. followed by incubation for 5 min at 95°C to inactivate reverse transcriptase.IFNG-AS1 and GAS5 in serum using specific primers supplied by Qiagen, ; for IFNG-AS1  and GAS5  and we used GAPDH primer  to standardize the expression pattern and quantify the target long non-coding RNAs  (Melting curve tests were carried out after the PCR cycles were completed to confirm the particular production of the expected PCR result. The cycle threshold (Ct) value is the number of qPCR cycles essential to the fluorescent signal to cross a specific threshold. By deducting the Ct values of GAPDH from those of the target long non-coding RNAs, Δquation)  to demonIFNG-AS1 and GAS5 and the clinical parameters, treatment, and response to treatment. The sensitivity and specificity of IFNG-AS1 and GAS5 each alone or combined regarding the discrimination between ITP cases and healthy control subjects the receiver operating characteristic (ROC) curve analysis was done. All the results of the tests were interpreted as significant by considering that p ≤ 0.05.Data was presented by mean ± SD (Standard Deviation), number and percentage, median and interquartile range; (IQR). SPSS version 22 (SPSS Inc) was used for analyzing data. The mean, SD, median, and range were calculated for the quantitative data. One-way ANOVA test was used for normal distribution data analysis in more than two groups. When variables were not normally distributed, the Mann–Whitney-U test (2 groups) or Kruskal Wallis test (more than two groups) was used in comparing groups. Otherwise, the independent-T test was used. The significance of the qualitative data was detected by chi-square (χ2). Pearson correlation was done to explore the association between The sample size equals 88 cases, to adapt the cost of research without decreasing the validity of results we used the formula;Necessary sample size = to get the appropriate sample size involved in this research, confidence levels 95% were converted into Z scores, the standard deviation was 0.5, and the margin of error was 0.05).IFNG-AS1 and GAS5 were at distinguishing between children within ITP and those within the control group and to explore the link between their levels and types of childhood ITP as well as with treatment regimens and response.A total of 88 ITP children, ranging in age from 1 to 13 years were included in this case-control study with mean ± SD = 4.9 ± 2.15 years and 49 (55.68%) patients were females and 88 healthy children with ages ranging from 2 to 12 years with mean ± SD = 5.1 ± 2.09 years and 45 (51.13%) were females. This study was constructed to see how effective the lncRNAs p = 0.254) and age (p = 0.172). Regards CBC picture, ITP patients had lower hemoglobin (HB) (mean ± SD = 9.9 ± 2.19 gm%), lower platelet count (PL) either before (mean ± SD = 9.33 ± 2.51× 103) or after treatment (mean ± SD = 129.14 ± 48.95 × 103) and higher absolute lymphocytic count (ALC)  than controls .Results represented in  showed tp = 0.095).There was no significant difference existed between the two groups regards white blood cell count (WBC)  with splenomegaly. All patients had no history of splenectomy, hepatitis B virus (HBV), or hepatitis C virus (HCV) infection. Treatment regimens and responses to treatment are shown in  were newly diagnosed <3 months, 27 (30.86%) with persistent ITP 3–12 months, and 19 (21.60%) had chronic >12 months ITP. Out of 88 patients; 5 (5.68%) with positive family history, 31 (35.23%) with history of bleeding, 59 (67.05%) with preceding febrile illness, 5 (5.68%) with shown in .IFNG-AS1  and GAS5  expressions in serum than that of healthy controls (p < 0.001 each) (Children with ITP had significantly higher 01 each) .IFNG-AS1 and GAS5 (p < 0.05) were present in persistent ITP (3–12 months) [ median (IQR) = 4.58 (0.31–22.39) for IFNG-AS1 and 3.77 (0.87–12.36) for GAS5] or chronic ITP (>12 months) [ median (IQR) = 5.6 (0.25–12.59) for IFNG-AS1 and 5.61 (1.15–16.91) for GAS5] when compared to newly diagnosed <3 months patients . Also, significant higher lncRNAs IFNG-AS1 and GAS5 were present in patients with partial response to treatment  or non-response [IFNG-AS1 median (IQR) = 4.19 (1.25–22.39) and GAS5 median (IQR) = 5.11 (2.34–15.27)] when compared to patients with complete response to treatment (IFNG-AS1 median (IQR) = 2.09 (0.2–14.58) and GAS5 (median (IQR) = 2.51 (0.09–10.33). As well, significant high IFNG-AS1 was detected in patients with positive family history (median (IQR) = 9.09 (2.54–22.39) than in those with negative family history (median (IQR) = 1.98 (0.2–8.04), p = 0.04), just significant p-value = 0.05 was detected between positive family history patients and negative family history patients regards GAS5.Regards the association between these genes and clinical characteristics, treatment and response to treatment in cases, we demonstrated that significant higher helicobacter infection, or treatment regimens   (Strong significant positive correlation was detected between < 0.001) , while nor GAS5) , 4. Theror GAS5) .IFNG-AS1 and GAS5 in ITP patients.IFNG-AS1 and GAS5 in ITP patients are demonstrating the diagnostic utility of these markers as predictors in distinguishing between patients with ITP and controls. LncRNA IFNG-AS1; AUC (95% CI) = 0.900 (0.769–0.980), p < 0.001, cut off point 1.38, sensitivity 66.3%, specificity 97.8%, total accuracy, 82.05%. Lnc RNA GAS5, AUC (95% CI) = 0.803 (0.625–0.975), p = 0.001, cut off point 2.58, sensitivity 68.5%, specificity 95.2%, total accuracy 81.85% and their combined expression AUC = 1.000, p < 0.001, sensitivity and specificity 100.0%.The ROC curves of lncRNAs After reviewing the previous literature to explore the functions of target genes in ITP pathogenesis, we designed  to show IFNG-AS1 and GAS5 in childhood ITP pathogenesis and their potential usage as targets of therapy. IFNG-AS1 is selectively expressed in T helper1 (Th1) cells by the effect of Stat4 and T-bet transcription factors (IFNG-AS1 promoting IFN-γ expression and secretion (+CD25+FoxP3+ Treg cell numbers (the major cells that relate to the maintenance of auto-tolerance)  . Normalilerance) , So, Kno for ITP .GAS5 is associated with the glucocorticoid receptors (GRs) through hindering binding of GRs to target genes’ glucocorticoid receptor elements (GREs), and repressed GRs transcriptional activity of endogenous glucocorticoid-responsive genes  is typically a benign self-limiting condition that resolves in a few months. However, between 20 and 30% of patients develop chronic ITP, which puts them at risk of serious complications including bleeding such as cerebral hemorrhage and blood cancer . Efficie therapy ,  of our patients was 5.1 ± 2·19 years, and there is no sex prediction unlike in adults, the initial platelet count was 9.33 ± 2.51×10t counts ,  = 3.08 (0.2–22.39), mean ± SD = 18.37 ± 19.54 for IFNG-AS1, and median (IQR) = 4.19 (0.9–16.91) for GAS5 than controls, Also, significant higher IFNG-AS1 and GAS5 were linked to patients with positive family history, patients with persistent or chronic ITP than those who are newly diagnosed, patients with no response or partial response to treatment when compared with patients who completely responded to treatment. In addition, IFNG-AS1 and GAS5 expression were significantly negatively correlated with platelet count after therapy.The most important clinically relevant findings were overexpressed lncRNAs IFNG-AS1, our results could be explained by the findings of Collier et al, 2012 who implied that IFNG-AS1 is a part of a larger family of lncRNAs called lincRNAs that positively control gene transcription. They defined IFNG-AS1 as a Th1-specific lincRNA that promotes the transcription and secretion of the IFN-γ (+CD25+FoxP3+ Treg levels (responsible for preserving self-tolerance by interaction with APC and lowering CD19+ B cell and CD8+ T cell responses)  ,  . Many piof Tregs ,  from active adult ITP patients was lower than in healthy controls. Furthermore, researchers discovered a positive correlation between IFN-γ and IFNG-AS1 transcript levels in PBMCs from healthy controls, as well as a similar change tendency after short-term activation. As a result, researchers speculated that IFNG-AS1 has been shown to enhance IFN-γ transcription. Furthermore, IFN-γ overexpression had a negative feedback effect on IFNG-AS1 expression, resulting in lower IFNG-AS1 levels in adult ITP patients  than in controls and they were positively linked  (Regarding tor (GR) .GAS5 is associated with glucocorticoid resistance through its direct attachment to the GR protein by competing with glucocorticoid receptor element (GRE) and acting as a decoy GRE, preventing glucocorticoid-induced gene transcription upregulation, and decreasing GCs activity so contributing to the development of numerous autoimmune diseases  are a powerful immunosuppressant and are often the essential treatment of inflammatory and autoimmune illnesses, along with preventing rejection in transplanted patients. Several studies indicated that diseases  .Support for this interpretation comes from controls , Also, patients . In a siGAS5 expression was downregulated in ITP patients’ PBMCs and ITP mice’s spleen tissues, and overexpression of GAS5 suppresses Th17 differentiation in vitro and relieved ITP in vivo via STAT3 reduction. Similarly, Li et al, 2019 concluded that the level of GAS5 was reduced in RA synovial tissues  were significantly lower than in healthy controls  The sample size of the current study was modest and selected from the same geographical area which provoked the possibility of selection bias. 2) Insufficiency of scientific literature demonstrating the precise function of target lncRNAs in general and in particular to pediatric diseases including childhood ITP. Further multicentric studies are needed to confirm our findings, verify the utility of target genes as targets of therapy, and better understand the molecular role of IFNG-AS1 and GAS5 are novel diagnostic and prognostic biomarkers for childhood ITP that can aid in a precise prediction of the disease’s progress at the time of diagnosis and could be a useful tool for treatment planning, reducing the risk of bleeding while avoiding drug side effects. We speculated that both genes have underlying molecular contributing roles in the development and prognosis of childhood ITP and may be used as targets of therapy to reduce the propagation of the disease to a chronic state.The current study contains several new and important insights; findings suggest that lncRNAs IFNG-AS1 and GAS5 are novel diagnostic and prognostic genetic markers for childhood ITP.LncRNAs


FABP4, FAS, FATP1 [SLC27A1], and LPL), adipokines (ADIPOQ and LEP) and autophagy-related genes  was detected by quantitative real-time polymerase chain reaction (RT-qPCR), and the protein expressions of PPARγ, ADIPOQ, LC3I, LC3II, Beclin1, and P62 were detected by western blot analysis. Compared with the control group, the knockdown of NNMT expression reduced lipid accumulation and triglyceride content in 3T3-L1 cells. The transcription of PPARγ, C/EBPα, SREBP1, FABP4, FASN, FATP1, LPL, Beclin1, ATG7, ATG12, and ATG14 decreased, while ADIPOQ and LEP transcription increased. The expression of PPARγ, LC3I/II, and Beclin1 proteins also decreased, while ADIPOQ and P62 protein expression increased. The over-expression NNMT group showed experimental results opposite to those described above. Interference with the expression of NNMT affects lipid accumulation, triglyceride content after cell differentiation, adipocyte differentiation-related transcription factors, genes related to lipid metabolism, the expression of adipokines, and autophagy in adipocytes.Nicotinamide N-methyltransferase (NNMT) is a methylase, and its expression is positively correlated with obesity and insulin resistance. This study aims to detect the effects of NNMT on lipid accumulation, triglyceride content, adipocyte differentiation-related transcription factors, genes related to lipid metabolism, adipokine expression, and autophagy in adipocytes. Lentivirus vectors and eukaryotic expression plasmids were used to interfere with NNMT expression. The Oil Red O method was used to detect lipid accumulation, and colorimetry was used to detect triglyceride levels. The transcription of adipocyte differentiation-related transcription factors , lipid metabolism-related genes (  This study found that knockdown of NNMT expression reduced lipid accumulation and triglyceride content in 3T3-L1 cells.Interference with the expression of NNMT also affects adipocyte differentiation-related transcription factors, genes related to lipid metabolism, the expression of adipokines, and autophagy in adipocytes.These results indicated that NNMT plays an essential role in these cellular processes.2, and childhood obesity is rising in developed and developing countries [Obesity has become a global issue, and its prevalence is increasing annually ,2. Worldountries . Obesityountries ,5. It isountries . Excessiountries . Moreoveountries . TherefoAdipose tissue is an endocrine organ and the main energy storage site in the human body. It secretes adipokines and hormones to maintain metabolic homeostasis . When nuNicotinamide N-methyltransferase (NNMT) is an enzyme that methylates nicotinamide to form N1-methylnicotinamide, which can be discharged from the human body. The expression of NNMT in human adipose tissue is positively correlated with obesity and insulin resistance ,13. StudAutophagy is an essential physiological process that provides nutrients to maintain important cellular function in nutrient deficiency while removing excessive or damaged organelles and misfolded proteins and lipids in the state of nutrient overabundance. Studies have shown that the autophagy level of adipose tissue in obese patients is upregulated, and the lack of autophagy in adipose tissue inhibits the formation of lipid droplets, thereby inhibiting fat accumulation . The infThis study aimed to detect the effects of NNMT on lipid accumulation, triglyceride content, adipocyte differentiation-related transcription factors, genes related to lipid metabolism, adipokine expression, and autophagy in adipocytes. We hypothesized that NNMT played a vital role in these cellular processes.2 and sufficient humidity. After the cells grew 70%–80%, they were detached with 0.25% trypsin to form a single cell suspension passaged in a ratio of 1:2–1:3.The 3T3-L1 cell line was purchased from the cell bank of the Chinese Academy of Sciences. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high-glucose medium containing 10% calf serum in an incubator at a constant temperature of 37°C with 5% COThe cells were inoculated into 6- or 12-well plates to achieve confluency of approximately 60% and then cultured  for two days to 100% confluence. The cells were cultured for another two days (day 0) after the solution change. Next, they were cultured in induction solution I  for another two days (day 2) before being cultured in induction medium II (DMEM containing 10% FBS with 10 μg/ml insulin) for two days (day 4), followed by DMEM containing 10% FBS. The medium was changed once every two days . The cells were collected for subsequent experiments according to the degree of cell induction and differentiation.2O) five times, and then photographed under a microscope. Next, 200 μL of isopropyl alcohol was added to each well; the cells oscillated on a shaker for 5 min. Finally, 200 μL of liquid was sucked into a 96-well plate and read at a 510 nm wavelength for the quantitative analysis.The Oil Red O Kit  was used to stain the 3T3-L1 adipocytes according to the manufacturer’s instructions. Differentiated and mature 3T3-L1 adipocytes were taken, fixed with 4% paraformaldehyde for 20 min after being washed, immersed in and washed with 60% isopropanol for 5 min, immersed in Oil Red O dye solution for 30 min, washed with deionized water  was reviewed using the National Center for Biotechnology Information’s (NCBI) gene database. Three loci  were selA lentivirus expression vector with element sequence hU6-MCS-CBh-gcGFP-IRES-puromycin and control insertion sequence TTCTCCGAACGTGTCACGT was obtained from Genechem. The virus and double-stranded shRNA underwent double-enzyme digestion and were ligated overnight at 16°C using T4 DNA ligase. Following this, the exchange reaction products were transformed into competent cells cultured upside down in Luria-Bertani Petri dishes. Colonies were selected for polymerase chain reaction (PCR), sequencing, and identification. The prepared plasmid and virus packaging auxiliary plasmids, pHelper1.0 and pHelper2.0, were co-transduced into 293 T cells, and the supernatant was collected for concentration and titer determination after 48 h.The 3T3-L1 cells were inoculated into 24-well plates and grown in DMEM containing 10% calf serum when the cells grew up to 40%–50% in the plate within 24 h. Then, 20 μL of HiTransG P infection-boosting solution (Genechem) and 10 μL of NNMT lentivirus interference vector with a multiplicity of infection (MOI) of 100 or 10 μL of control vector were added to the cells after thorough mixing to achieve the required volume of 500 μL per well. The cell status was observed following overnight incubation, and the culture medium was replaced with fresh medium. Then, 48–72 h after infection, the expression of green fluorescent protein was observed under a fluorescence microscope. The downstream experiment was conducted if the fluorescence rate was greater than 80%. Finally, the cells were subcultured to induce differentiation for subsequent experiments.The 3T3-L1 cells were inoculated into 6-well plates and grown in DMEM containing 10% calf serum to achieve cell confluency of 80% at transfection. Next, 2 μg of the control plasmid and the NNMT overexpression plasmid  were added to 200 μL of jetPRIME buffer (Polyplus-transfection), vortexed for 10s, and then centrifuged. Following this, 4 μL of jetPRIME transfection reagent (Polyplus-transfection) was added, vortexed for one second, and incubated at room temperature for 10 min. This solution was then added to the cell culture medium. The culture medium was replaced with fresh medium every 24 h for subsequent experiments.6–5 × 106 cells were collected, and 1 ml of extractive buffer was added to the cells. The cells were crushed by ultrasonic waves at 2 s intervals at 20% power for one minute. The samples were then centrifuged at 8,000 g for 10 min at 4°C, and the supernatant was taken. Next, 40 μL of supernatant, standard, and blank control (distilled and ddH2O) samples were collected, and 125 μL of extractive buffer was added to each sample and mixed thoroughly. Reagent I was added, shaken vigorously for 30s, and allowed to stand for 5 min; this process was repeated three times to obtain 15 μL of upper-layer solution. Then, 50 μL of reagent II and 15 μL of reagent III were added, and the mixture was treated in a water bath at 65°C for 3 min. Next, 50 μL of reagent IV and 50 μL of reagent V were added, and the mixture was treated in a water bath at 65°C for 15 min. After cooling, 100 μL of the medium was pipetted into a 96-well plate to determine the absorbance at 420 nm. The triglyceride content was measured using the calculation /, and the results were normalized for the protein concentration.A triglyceride colorimetric assay kit was obtained from Abbkine (China). According to the manufacturer’s instructions, 4 × 10–ΔΔCt. The primer sequences are detailed in The cells were lysed with RNAiso Plus ; the RNA was extracted according to the manufacturer’s instructions and then reverse-transcribed into complementary (cDNA) . Then, real-time quantitative PCR (RT-qPCR) was performed using the RT-qPCR kit SYBR Green Master Mix  and an RT-qPCR instrument . The reaction conditions were as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 10s and 60°C for 30s, and then another stage at 95°C for 15s, 60°C for 60s, and 95°C for 15s. The data obtained from three independent experiments were analyzed using the formula RQ = 2The cells’ total protein content was extracted using RIPA lysis buffer , and the protein concentration  was detected via the BCA method. The total protein loading was 20 μg; it was subjected to electrophoresis on 10% or 12.5% SDS-PAGE gel  and transferred to a 0.22 μm PVDF membrane . It was incubated with 5% nonfat dried milk (blocking buffer) in the shaker for 1 h, and the primary antibody was incubated overnight at 4°C. The primary antibodies were anti-PPARγ , anti-adiponectin , ɑ-tubulin , β-actin , GAPDH , anti-NNMT , anti-LC3 , Beclin 1 , and P62 . The secondary antibodies were diluted with PBST and incubated for 1 h at room temperature with goat anti-rabbit IgG horseradish peroxidase  or goat anti-mouse IgG horseradish peroxidase . The reactive bands were detected by an enhanced chemiluminescence kit , after which the Image-Pro Plus gray-level analysis software was used to analyze the gray-level value of the displayed protein band. The sample’s relative expression was calculated using the expression of the internal reference protein as a control.The SPSS 18.0 software was used for the analysis, and the data were expressed as mean ± standard deviation. An analysis of variance or independent sample t-test was used to determine statistical differences, and P < 0.05 was considered statistically significant.FABP4, FASN, FATP1, LPL, Beclin1, ATG7, ATG12, and ATG14 decreased, and ADIPOQ and LEP transcription increased. The expression of PPARγ, LC3I/II, and Beclin1 proteins also decreased, while ADIPOQ and P62 protein expression increased. The over-expression NNMT group showed experimental results opposite to those described above. These results indicated that NNMT played a vital role in these cellular processes.This study found that knockdown of NNMT expression reduced lipid accumulation and triglyceride content in 3T3-L1 cells. Meanwhile, the transcription of PPARγ, C/EBPα, SREBP1, Beclin1, ATG7, ATG12, and ATG14 . As shown in nd ATG14 . The mosFABP4, FASN, SLC27A1, and LPL, significantly decreased, and the mRNA levels of adipokine ADIPOQ and LEP significantly increased . The pcegulated . The PPAegulated .Figure Beclin1, ATG7, ATG12, and ATG14 (Beclin1, ATG7, ATG12, and ATG14 significantly increased (As mentioned previously, we found that the NNMT and autophagy-related genes increased during the induction and differentiation of the 3T3-L1 cells. To investigate whether NNMT could influence autophagy-related genes in 3T3-L1 cells, LV-NNMT-RNAi3 and pcDNA3.1-NNMT-3xFlag-C were transduced into the cells and induced to differentiate. The change in the autophagy-related genes on day 6 was recorded. Compared with the control group, the knockdown of NNMT expression resulted in a significant decrease in the mRNA levels of nd ATG14 . In addind ATG14 . LC3 is ncreased , the proncreased . These rThe rising prevalence of obesity is a global health issue that leads to an increased risk of disease . AccordiAtg7 in 3T3-L1 preadipocytes inhibits lipid accumulation and decreases the protein levels of adipocyte differentiation factors [Atg5 or pharmacological inhibition of autophagy had similar effects [Atg7 generated lean mice with decreased WAT mass and enhanced insulin sensitivity [The differentiation and lipid accumulation of adipocytes are closely related to the occurrence and development of obesity . In a st factors . Knockdo effects . An adipsitivity . Some stsitivity . Shin etsitivity . Nicotinsitivity . High nesitivity . Both basitivity ,29. In tThe effect of NNMT on adipocyte differentiation-related transcription factors PPARγ, C/EBPα, and SREBP1 was explored in this study. A ligand-activated transcription factor, PPARγ’s expression is highest in adipocytes, plays a key role in lipogenesis, energy balance, and lipid biosynthesis, and is also involved in lipoprotein metabolism and insulin sensitivity ,31. ConsHighly conservative members of the basic leucine zipper protein family, C/EBPα, are an important transcription factor involved in regulating adipogenic differentiation. Research has shown that C/EBPα knockout mice display metabolic abnormalities and cannot complete lipid accumulation in the adipocytes . A transFABP4, FASN, SLC27A1, and LPL and adipokines ADIPOQ and LEP. As one of the most abundant proteins in adipocytes, through interaction with hormone-sensitive lipase and PPARγ, FABP4 maintains lipocyte homeostasis and regulates lipolysis and lipogenesis [de novo synthesis of intracellular fatty acids, FASN can catalyze the synthesis of long-chain fatty acids from acetyl-CoA [SLC27A1 is involved in the uptake and metabolism of long-chain fatty acids. Studies have shown that after the knockout of the SLC27A1 gene in mice, the fatty acid uptake in the adipocytes decreases significantly; thus, it can control diet-induced obesity and insulin resistance [FABP4, FASN, SLC27A1, and LPL, thereby affecting lipid accumulation. Moreover, it results in changes in the transcriptional activities of adipokines ADIPOQ and LEP, which also affects lipid accumulation.We also explored the effect of NNMT on lipid metabolism-related genes ogenesis . Geneticogenesis . A key eetyl-CoA . As one sistance . Next, Lsistance . A protesistance . We founFABP4, FASN, FATP1, and LPL genes related to lipid metabolism were downregulated, while the transcription of ADIPOQ and LEP and the expression of the ADIPOQ protein increased. Therefore, we believe that NNMT may regulate the expression of these genes in lipid accumulation. In addition, the transcription of autophagy-related genes Beclin1, ATG7, ATG12, and ATG14 was decreased along with the protein expression ratio of LC3II/I and the protein expression of Beclin 1. Moreover, the increased expression of P62 compared with the control group indicated reduced levels of autophagy. Thus, we think that the mechanism of NNMT in lipid accumulation may also include autophagy regulation. An inverse result was observed in the overexpression NNMT group. The above results indicated that interference with NNMT affects lipid accumulation in 3T3-L1 cells, possibly by interfering with the transcription and expression of PPARγ, C/EBPα, and SREBP1 and affecting the functions of FABP4, FAS, FATP1, and LPL, which might be related to autophagy.To investigate the effects of NNMT on adipocyte function and the possible mechanisms of its action, we used lentiviral knockdown and overexpression plasmids to interfere with – and then induce – differentiation. On day 6, we observed a significantly decreased number of cells with lipids. Smaller and fewer lipid droplets formed in the 3T3-L1 cells, decreasing the fat accumulation and triglyceride content in the NNMT knockout group. Moreover, the transcription of adipocyte differentiation-related transcription factors PPARγ, C/EBPα, and SREBP1 decreased, as did the expression of PPARγ protein. The transcription of the N1-methylnicotinamide, a metabolite of NNMT, was found to be able to inhibit reactive oxygen species, inflammation, apoptosis in palmitic acid-treated H9C2 cells and improve inflammation, apoptosis, and fibrosis damage in free fatty acids-bound-albumin-overloaded mice.  In our sThis study had some limitations. First, we didn’t analyze the potential influence of different genders. Furthermore, the effect of NNMT inhibition in fully differentiated cells was also not investigated. Future studies would focus on the effects of N1-methylnicotinamide and the substrate of NNMT on adipocytes. Whether the obese phenotype was able to be reversed when adipocytes were already present and loaded with lipids also deserves to be investigated.FABP4, FASN, FATP1, LPL, Beclin1, ATG7, ATG12, and ATG14 decreased, and ADIPOQ and LEP transcription increased. The expression of PPARγ, LC3I/II, and Beclin1 proteins also decreased, while ADIPOQ and P62 protein expression increased. The over-expression NNMT group showed experimental results opposite to those described above. These results indicated that NNMT played an essential role in these cellular processes.In conclusion, knockdown of NNMT expression reduced lipid accumulation and triglyceride content in 3T3-L1 cells. Meanwhile, the transcription of PPARγ, C/EBPα, SREBP1,


Thetetramates were found to release chelated metal cations acquired duringchromatographic purification by mild acid wash. Some compounds inthe library showed good antibacterial activity against Gram-positivebacteria. Cheminformatic analysis demonstrates that the most activecompounds were Ro5-compliant and occupy a narrow region of chemicalspace, distinct from that occupied by other known antibiotics, withthe most potent compounds having 399 < Mw < 530 Da;3.5 < cLogP < 6.6; 594 < MSA <818 Å2; 9.6 < rel. PSA <13.3%. MIC values were shifted tohigher concentrations when tested in the presence of HSA or blood,but was not completely abolished, consistent with a plasma proteinbinding (PPB) effect.A general route, which provides direct access to substitutedbicyclictetramates, making use of Dieckmann cyclization of oxazolidines derivedfrom  Of interest was whether phenylserines couldbe used for similar cyclizations, especially so since such systemswould also further expand the substrate scope by enabling greaterfunctional group diversity around the bicyclic ring    (aq) solution andthe solution left to stir for 10 min. Benzaldehyde (2.1 eq) was thenadded at room temperature to form the initial aldol product B, which led to the formation of a condensation cake thatwas left for 3 days at room temperature. The thus obtained aldol product 4a was then subsequently treated with 3M HCl (aq) to formβ-aryl serine 3a along with regenerated benzaldehyde.The excess aldehyde could be removed from the desired β-arylserine 3a by addition of Et2O and water, andseparation of the layers followed by evaporation of the aqueous layerto dryness in vacuo gave β-aryl serine 3a in highyield and good diastereoselectivity, along with NaCl. Of greatestvalue to us was this high stereoselectivity, rather than yield andpurity, and crude material was taken forward in the synthetic sequence.We took inspiration from Erlenmeyer’sreportreaction 1.8 The caldehyde1. This saldehyde1. The foroduct 4 1. Moreoventry 5, 1; howeveentry 4, 1. Thus, entry 4, 1, and soentry 4, 1 found f3e did not form in significantquantities and mostly unreacted glycine was obtained  and Et3N at rt overnight (1H NMR data for trans-oxazolidinone 6 were consistent with literature NMR data (threo-derived oxazolidinones10 and erythro-derived oxazolidinones11), confirming that the amino acid 3a formed was indeedthe threo isomer (Table S1).With β-aryl serines t yields 2.9 In orvernight 2. The 1H14 amino esters 5a–e were reactedwith pivaldehydein the presence of Et3N and petroleum ether 40:60, at 100°C for 16–24 h to form oxazolidines 7a–e in good yields as roughly a 1:1 mixture of diastereomers at theC2 center, as determined from 1H NMR spectroscopic studiesof the crude material . Importantly, the relative stereochemistryof oxazolidines 7a–e at positions C4 and C5 wasconserved from the starting amino esters 5a–e.Using literature precedent,material 2. That t15 Asthe crude oxazolidines 7a–e were relatively pureby NMR analysis, they were therefore used directly and N-acylated using ethyl hydrogen malonate under DCC/DMAP coupling conditionsto furnish the malonamide products, although in poor yields. Applicationof more forcing conditions, by reaction with ethyl malonyl chlorideunder basic conditions, gave malonamides 8a–e and 9a-e in better yields—typically between 60 and 88%—asapproximately a 3:2 mixture of diastereomers at the C2 center (Table S3). These were obtained as stable oilsbut were extremely difficult to separate by chromatography.An attempt to purify these oxazolidines by chromatographic purificationgave low recovery of the product, and this is likely to arise fromretro-aldol reactions for oxazolidines with C2 aromatic substituents,which have been reported previously.cis-malonamides 8b–d was assigned using nOe and NOESY spectroscopicstudies , and confirmedby single crystal X-ray diffraction of the racemic ethyl ester analogue 8f (Table S3).16 In the case of 2,4-trans-malonamides 9a–d, low temperatureVT ROESY experiments were used to determine their relative stereochemistry; this is further discussedbelow. Importantly, there was no evidence of any epimerization fromthe starting relative configuration at the C4 and C5 positions ofmalonamides 8a–e and 9a–e underthe basic conditions of the N-acylation, and overall,the N-acylation of oxazolidines 7a–e was not selective, unlike similar reactions of oxa(or thia)zolidinesderived from serine, threonine, and cysteine,5 suggesting no substantial energy difference between malonamides 8a–e and 9a–e.The relative stereochemistry of 2,4-studies S1, and c8a–e with 9a–e. Malonamides 8a–e showed sharp NMR peaks atroom temperature, whereasmalonamides 9a–e had very broad NMR signals , to the point where key signals disappeared,and this was attributed to fluxional ring conformational effects.For the former, all substituents may reside in pseudo-equatorial positionsgiving a clear-cut energy minimum, but for the latter, at least onemust reside in a pseudo-axial position, giving several energeticallylow-lying structures which are each visible and averaged on the NMRtime-scale. Low temperature VT-NMR experiments slightly sharpenedthe NMR peaks .Of particular interest was the contrasting NMRbehavior of malonamides 8a–e and 9a-e were reacted directly with KOtBu in THF overnight at reflux. However, poor yieldswere obtained for the product tetramates 10a–e–typically between 30 and 40%—along with other unidentifiedside products. In order to understand why such poor yields of tetramatesarose, a small amount of each malonamide diastereomer was separatedby careful chromatographic purification and each reacted under Dieckmannconditions individually with KOtBu inTHF overnight at reflux. Of interest is that malonamides 8a–e proved to be resistant to the Dieckmann cyclization ; these tetramates 10a–e werefound to be unstable to chromatographic purification, and this issimilar to that observed by Andrews et al. for analogous C7-ethylester tetramates derived from l-serine.4 They were also unstable in CDCl3 solution, wherethey rapidly degraded to C7-decarboxylated tetramates 11a–e—as determined from NMR spectroscopic and mass spectrometricstudies  . Of note is that malonamide 8a was more stable than malonamide 9a by approximately4.1 kcal/mol (17.2 kJ/mol), accounting for its experimentally observedslight diastereomeric preference —so that formation of the latteris strongly disfavored; this was in agreement with the experimentallyobserved chemoselectivity . The next highest energy transitionstates were enolate-8a, which had an Ea = 20.3 kcal/mol (84.9 kJ/mol), and enolate-8a′, which had an Ea = 19.5 kcal/mol (81.6 kJ/mol), suggestingthat enolate-9a was the kinetically more favored reactionpathway leading toward the formation of tetramate 10a , and consistent with experiment. Moreover, tetramate 10a was considerably more stable than tetramates 15 and 13, by 12.1 (50.6 kJ/mol) and 9.3 kcal/mol (38.9 kJ/mol),respectively. Overall, this suggested that tetramate 10a–e was both the kinetic and thermodynamic product. This outcome likelyarises from the steric effect of the t-butyl group,which while simultaneously protecting the O and N groups, also providesa significant steric bias to influence reaction chemoselectivity andtherefore direction of ring closure.In order to understand this process in moredetail, DFT calculationswere conducted invoking all the intermediates and transition statesalong with solvent (THF) and base .Interestingly, these tetramates were far more stable than their ethylester precursors 10a–e, fully surviving chromatographicpurification and storage in CDCl3 solution for many months.However, upon chromatography on silica gel, metal chelate formationwas rapid, readily seen in the NMR spectrum, where broad peaks wereobserved; earlier work has shown that these are likely to be principallydivalent metal cations, including Mg2+ and Ca2+, along with monovalent cations, such as Na+ and K+.17 For example, in the case ofcompound 12a ,the NMR peaks for H4 and H5 were extremely broad after chromatographicpurification, consistent with their proximity to the metal chelationsite, whereas the tBu, aromatic, and adamantylgroups were relatively sharper, being further away from the metalchelation site. However, when metal-chelated C7-carboxamides werewashed with 10% citric acid solution, the NMR peaks significantlysharpened. The fact that only such a mild acid wash was needed, incomparison to the reported 2M HCl (aq) which is usually required,suggested that the C4-aromatic substituents exerted steric disruptionof the metal chelation across the tricarbonyl core.17 Additionally, C7-carboxamides were found to exist as amixture of tautomeric pairs AB/CD as observed by NMR spectroscopy.The chemoselective Dieckmanncyclization of malonamides ound 12a S5,the NFigure S1), and itwas clear that there appeared to be no epimerization after aminolysisat any of the stereogenic centers within the bicyclic molecule. Furthermore,a single crystal X-ray diffraction structure of compound 12a confirmed the relative stereochemistry , itwas assumed that all C7-carboxamidotetramic acids had the same relativestereochemistry.nOe and NOESY studies were conducted to determinethe relativestereochemistry of these C7-carboxamidotetramic acids, giving thetypical correlations indicated S1, and iTable S6) against two example organisms,methicillin-resistant Staphylococcus aureus (MRSA) and E. coli. C7-Ethyl estertetramates derived from the methyl esters of l-serine, l-threonine, and l-cysteine or unfunctionalized/simpletetramic acids have been observed to have no intrinsic antibacterialactivity against Gram (+) and Gram (−) bacteria, with MIC valuesin most cases exceeding 250 μg/mL.5 In keeping with these observations, tetramates 10a,b showed no activity against MRSA and E. coli, but of interest was some modest activity for compounds 10c–e against MRSA with MIC values ranging between 15.6 and 31.3 μg/mL.By contrast, upon conversion of the C7-ethyl ester to a C7-aromaticcarboxamide, good to excellent antibacterial activity was observedagainst MRSA, with activity ranging between 0.49 and 15.6 μg/mL,although no activity was observed against E. coli (Table S6). Although 12b exhibited only modest antibacterial activity (MIC = 7.80 μg/mL),additional substituents around the aromatic carboxamide gave muchbetter activities, for example, p-Br 12c and p-N O 212d had a MICvalue of 3.90 μg/mL; p-CF312e and p-cyclohexyl 12s hadMIC = 1.00 μg/mL; and p-hexyl 12m had MIC = 0.49 μg/mL, suggesting that a hydrophobic tail wasimportant. Ortho-and para-Substitutedaromatic rings 12g–12l were well-tolerated, withactivity ranging between 0.49 and 3.90 μg/mL, and fluoro-substituted 12i and 12l had high activities. In the caseof aliphatic carboxamides, more hydrophobic side chains at the C7-positiongave better activity against MRSA, and dodecyl 12r andnonyl 12q were far more active (MIC = 0.49 μg/mL)than hexyl 12p (MIC = 3.90 μg/mL). While t-Bu 12v had comparable activity with the hexyl 12p, benzyl 12u only exhibited modest bioactivity(15.60 μg/mL). By contrast, a more polar substituent at theC7-position (pyran 12t) compromised the antibacterialactivity (125 μg/mL). This was consistent with reports by Panduwawalaet al.17 and Jeong et al.18 that polar substituents at the C7-position significantlyworsened antibacterial activity for bicyclic tetramates. An exceptionwas 12d, which had good bioactivity. Although cyclohexyl 12n and cycloheptyl 12o had a MIC value of 1.00μg/mL, they were not as potent as the adamantyl substituent 12a (MIC = 0.25 μg/mL), this being the most potent compound.Since carboxamide 12a was the most active compound, theC4-aromatic substituents in the tetramate core were varied; for 13a and 15, both the p-Me substituent(MIC = 3.90 μg/mL) and p-Cl substituent (MIC= 7.80 μg/mL) had a detrimental impact on the antibacterialactivity, although p-F 14and p-Br 16  only had a slightly reduced bioactivity in comparisonto 12a. Compound 13b had the same activityas 12g at 1.00 μg/mL. Overall, increasing lipophilicitycorresponded to higher potency against MRSA.With access to these derivativesroutinely available, an assessment of their antibacterial activitywas made by measuring minimum inhibitory concentration (MIC) values(10 and amides 12–16 (Table S6) shows that the library is characterized by 345 <Mw < 529 Da 1.7 < cLogP < 6.6;9.6 < rel-PSA < 20.6%, and many of the bioactive compounds arewithin the scope of Ro5   5.50Hela cell line/MIC(S. aureus)] , andbetter than the desired >10-fold higher antibacterial activityovercytotoxicity.19The selectivity of these compounds for activityagainst prokaryoticcells over mammalian cells was analyzed by considering their cytotoxicityagainst Hela, heK-293, CaCo, and MDCK human cell lines, allowing estimationof the therapeutic ratio [ECaureus)] 3. While12–16 were testedin the presence of blood, the antibacterial activity for most of thesecompounds significantly reduced, with 15 out of 26 carboxamides onlyshowing activity at concentrations >124 μg/mL. Nonetheless,11 carboxamides were active at concentrations between 31.25 and 62.5μg/mL,an important outcome, as earlier findings had demonstrated that C3/C7-acyl/carboxamidotetramatesin most cases suffered from either partial or complete loss of efficacyunder similar conditions.5 As tetramicacids are well-known metal chelators,20 of interestwas to investigate whether this loss of activity was due to metalchelation or plasma protein binding (PPB), specifically with humanserum albumin (HSA).18 To answer this question, bicyclictetramate 12a was tested against MRSA using a broth dilutionassay, under standard conditions and with the addition of similarmetal cations  with concentration as that foundin blood, respectively. The activity was found to be slightly improvedfor the latter {0.49 vs 0.24 [MIC (μg/mL) against MRSA]}, suggestingthat the weaker metal chelating properties of these aryl-substitutedtetramates discussed above may be important for in vivo activity intherapeutic application. Bicyclic tetramate 12a was alsotested against MRSA using a broth dilution assay, under standard conditionsbut in the presence of one-third the serum albumin concentration asthat found in human blood. The activity dropped 16-fold {7.81 [MIC (μg/mL) against MRSA]}, and this outcome is similarto the work by Panduwawala et al. and Jeong et al.,18 who had also earlier demonstrated that carboxamidotetramic acidslost antibacterial activity in the presence of HSA. A review has shownthat PPB strongly correlates with increased hydrophobicity, and thismight point to a problem with the tetramate class, which clearly dependsupon hydrophobicity for their antibacterial activity, as noted above.21However, when carboxamides threo-arylserines, has been developed; thisrequired the development of a diastereoselective route to β-hydroxy-β-aryl-α-aminoacids, achieved by an aldol-like reaction of glycine with substitutedbenzaldehydes under alkaline conditions. The reactions of bicycliclactams24 are governed by several phenomena including steric,26 electronic,27 and hydrogen bonding28 effects, and the work described here shows thatring substituents may critically influence reaction outcomes leadingto such functionally dense bicyclic ring systems, likely to be ofrelevance to the recently identified clausenamide group of naturalproducts.29 This work expands opportunitiesfor scaffold hopping from tetramates to pyroglutamates, by providinga general route to highly substituted systems.31 For the synthesized N,O-bicyclic tetramates, ithas been demonstrated that C7-ethyl ester tetramates 10a–e showed either no antibacterial activity or only weak antibacterialactivity against MRSA. However, C7-carboxamides 12–22 showed much improved antibacterial activity with large hydrophobiccarboxamide pendants at the C7-position giving the most potent antibacterialactivity. However, the use of a more polar substituent at the C7-positioncompromised bioactivity. All the compounds screened showed no antibacterialactivity against E. coli. The C7-adamantylcarboxamidotetramate 12a was very selective for bacteria over mammalian cells.Many of the compounds synthesized were Ro5-compliant and occupieda distinct chemical property space different from that occupied byother known antibiotics, with the most potent compounds having 399< Mw < 530 Da; 3.5 < cLogP < 6.6; 594 < MSA <818 Å2 9.6 < rel. PSA <13.3%. Unfortunately, MIC values wereshifted to higher concentrations when tested in the presence of HSAor blood, consistent with a plasma protein binding (PPB) effect, eventhough metal chelation was reduced in the more densely functionalizedtetramate system. Considering the physicochemical properties, potency,and toxicity of these compounds, the most promising candidates forfurther optimization are 12a and 12i, alongwith 22 even though it was of high molecular weight (Mw > 500 g/mol).A general route which provides direct accessto substituted bicyclictetramates, making use of Dieckmann cyclization of oxazolidines derivedfrom


Colletotrichum scovillei causes severe anthracnose disease on the fruit of sweet pepper and chili pepper (Capsicum annuum L.) worldwide. Understanding the biology of C. scovillei would improve the management of fruit anthracnose diseases. The cyclic adenosine monophosphate (cAMP) signaling pathway regulates diverse cellular and physiological processes in several foliar fungal pathogens. We investigated the roles of the cAMP signaling pathway in C. scovillei using pharmaceutical and genetic approaches. Exogenous cAMP was found to increase conidiation, appressorium formation, and anthracnose disease development in C. scovillei. CsAc1, CsCap1, and CsPdeH, which regulate the intracellular cAMP level, were deleted by homology-dependent gene replacement. Expectedly, the intracellular cAMP level was significantly decreased in ΔCsac1 and ΔCscap1 but increased in ΔCspdeh. All three deletion mutants exhibited serious defects in multiple fungal developments and pathogenicity, suggesting regulation of the intracellular cAMP level is important for C. scovillei. Notably, exogenous cAMP recovered the defect of ΔCsac1 in appressorium development, but not penetration, which was further recovered by adding CaCl2. This result suggests that CsAc1 is associated with both the cAMP and Ca2+ signaling pathways in C. scovillei. ΔCscap1 produced morphologically abnormal conidia with reduced tolerance to thermal stress. ΔCspdeh was completely defective in conidiation in C. scovillei, unlike other foliar pathogens. Taken together, these results demonstrate the importance of cAMP signaling in anthracnose disease caused by C. scovillei.The ascomycete fungus  Fungal plant pathogens have evolved diverse and sophisticated signal transduction systems for dispersal and survival in response to the environment and host . PerceptMagnaporthe oryzae, deletion of adenylyl cyclase-encoding MAC1 caused pleiotropic defects, including reduced vegetative growth and conidiation, delayed conidial germination, and complete loss of appressorium formation  in tropical and subtropical regions  and its transformants were routinely grown on V8 juice agar medium (V8A), potato dextrose agar medium (PDA), and oatmeal agar medium (OMA)  and National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Sequences were aligned using MEGA X and illustrated using BioEdit 7.2.The wild-type um (OMA) . Myceliaum (OMA) . Sequenchygromycin B phosphotransferase (HPH) was fused with upstream and downstream segments of each gene by rounds of fusion PCR with corresponding primers 5F/3R (via Southern blotting. The complemented strains were generated by co-introducing the geneticin-resistant cassette and segments amplified with primers NF/NR (A modified double-joint PCR was used to fuse DNA segments for generation of target deletion mutants as previously described . DNA fravia a quick extraction method used for screening PCR and a standard method used for Southern blotting (β-tubulin (CAP_007327) was used as a reference gene in RT-PCR and qRT-PCR. The qRT-PCR was performed using the StepOne real-time PCR system  with two replicates in three independent experiments. The relative gene expression was calculated as 2−ΔΔCT  and then powdered with liquid nitrogen. The intracellular concentrations of cAMP were measured using a cAMP complete ELISA kit  according to manufacturer’s instructions. For each sample, 0.1 g of fine powder were homogenized in 1 mL of 0.1 M HCl. Intracellular concentrations of cAMP were measured in three independent experiments with three replicates.2O. To distinguish conidiophores, lactophenol aniline blue solution was used to stained mycelia (4/mL), harvested from 7-day-old OMA, on the hydrophobic surface of coverslips and the hydrophilic surface of slid glasses , and the surface of pepper fruits. The percentage of appressorium formation were calculated by counting the number of appressorium-forming conidia in a total of 100 conidia. All data were collected from three independent experiments with triplicates per experiment.Mycelial growth was evaluated by measuring colony diameters on CMA and MMA at 5 days. Hyphae grown on MMA were stained with calcofluor white to visualize septa under a fluorescent microscope. Conidiation was evaluated by counting the number of conidia in 7-day-old V8 agar with 5 mL distilled H mycelia . Appress4/mL), harvested from 7-day-old OMA, were inoculated onto intact and wounded pepper fruits, and incubated in humid plastic boxes at 25°C. Lesion sizes of anthracnose disease on pepper fruits were measured by using Image J. To observe infectious hyphae, thin sections were sliced using a razor blade from infected pepper fruits and then immersed in mixture of acetic acid, chloroform, and methanol , CsCap1 (CAP_009109), and CsPdeH (CAP_005557), which encode Ac1, Cap1, and PdeH, respectively, known for modulating intracellular cAMP concentration , Cap1 (XP_003714719.1), and PdeH (XP_003710562.1). Phylogenetic analysis showed that CsAc1, CsCap1, and CsPdeH were closely related to orthologs in filamentous fungi, and distantly related to orthologs in yeasts  and ΔCscap1 (540.6 ± 75.6 fmol/mg), but markedly higher in ΔCspdeh (2957.2 ± 133.9 fmol/mg), compared to the wild-type (831.8 ± 150.8 fmol/mg). This result suggests that CsAc1, CsCap1, and CsPdeH modulate the intracellular cAMP concentration of C. scovillei.To investigate roles the of CM broth Figure 2CsAc1, CsCap1, and CsPdeH are involved in mycelial growth, colony diameters on CMA and MMA were measured. The colony diameter was significantly smaller in ΔCsac1 , ΔCscap1 , and ΔCspdeh , compared to the wild-type  (ΔCsac1 (14.7 ± 1.5 µm), ΔCscap1 (14.3 ± 2.8 µm), and ΔCspdeh (16.8 ± 2.4 µm), compared to the wild-type (38.6 ± 1.4 µm)  × 104 conidia/mL] and ΔCscap1 [(17.9 ± 4.3) × 104 conidia/mL] compared to the wild-type [(66.4 ± 3.1) × 104 conidia/mL]  than the wild-type (97.5 ± 0.8%)  and ΔCscap1 (84.3 ± 9.0%)  resulted in defects in appressorium formation in M. oryzae . In M. oryzae, deletion of MAC1, Cap1, or PdeH severely reduced invasive hyphal growth  funded by the Ministry of Education, Science and Technology, and by a grant (918019043HD020) from the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Furthermore, the 14Ser/10-10Ser vesicles were only found as single aggregates  in a very narrow compositional range and as a bimodal population (average diameters of 80 and 300 nm). In contrast, the 12Ser/8-8Ser vesicles were found for a wider sample compositional range and as unimodal or bimodal populations, depending on the mixing ratio. The aggregate size, pH and zeta potential of the mixtures were further investigated. The unimodal 12Ser/8-8Ser vesicles  are particularly promising for application as drug/gene nanocarriers. Both chain length asymmetry and total length play a key role in the aggregation features of the two systems. Molecular insights are provided by the main findings.Mixtures of cationic and anionic surfactants often originate bilayer structures, such as vesicles and lamellar liquid crystals, that can be explored as model membranes for fundamental studies or as drug and gene nanocarriers. Here, we investigated the aggregation properties of two catanionic mixtures containing biomimetic surfactants derived from serine. The mixtures are designated as 12Ser/8-8Ser and 14Ser/10-10Ser, where mSer is a cationic, single-chained surfactant and n-nSer is an anionic, double-chained one . Our goal was to investigate the effects of total chain length and chain length asymmetry of the catanionic pair on the formation of catanionic vesicles, the vesicle properties and the vesicle/micelle transitions. Ocular observations, surface tension measurements, video-enhanced light microscopy, cryogenic scanning electron microscopy, dynamic and electrophoretic light scattering were used to monitor the self-assembly process and the aggregate properties. Catanionic vesicles were indeed found in both systems for molar fractions of cationic surfactant ≥0.40, always possessing positive zeta potentials ( Model membrane systems, like liposomes, lamellar liquid crystals, supported lipid bilayers and Langmuir monolayers, have been extremely relevant for fundamental studies related to the physiological and biophysical properties of biological membranes ,2,3. TheAmong the colloidal systems prone to the formation of model bilayers are the so-called catanionic mixtures ,13,14. TThe wide diversity of commercially available surfactants has provided a solid basis to generate many catanionic systems and catanionic vesicles, leading to the performance of extensive fundamental studies ,25,26,27In particular, we previously reported that serine residues, conveniently made as cationic or anionic polar headgroups, offer a good toxicological profile for surfactants ,56,59,60N,N-dioctylserinate (3a) in one step with 95% yield. On the other hand, reductive amination of decanal afforded the N-monoalkylated compound (A), which was reacted again with decanal to yield the N,N-didecylserine methyl ester (3b). The anionic surfactants 4a/4b were readily accessed by saponification of the corresponding N,N-dialkylserine methyl esters (3a/3b) with KOH/MeOH. The serine-based surfactants were synthesized according to procedures previously developed by our group ,59. The N-alkylated precursor (5c/5d) by reductive amination, using dodecanal or tetradecanal. Exhaustive methylation of these compounds, followed by removal of the tert-butyl protecting group yielded the target single chained surfactants 6c/6d. For the synthesis of the cationic surfactants, the serine derivative 2 was used as starting material. The first step in the reaction sequence consisted in the transformation of 2 into the corresponding 1H/13C NMR spectroscopy and mass spectrometry data, which is in accordance with previous reports [All the compounds were identified by  reports ,59. SpecN,N-didecylserinate (4b): white powder (82%). 1H NMR : δ 4.12 OH), 4.03 , 3.85 OH), 3.35–3.20 ), 1.83–1.67 ), 1.44–1.23 m, 28H, 2 x (-CH2-)7), 0.90 . 13C NMR : δ 170.9 (C=O), 69.2 (-CH), 59.7 (-CH2OH), 53.7 (-N-CH2-), {33.1, 30.6(4), 30.5(6), 30.4(6), 30.2, 27.7, 25.8, 23.8 (-CH2-)8)} and 14.5 (-CH3).Potassium ® ultrapure water followed by thorough mixing of all the components by vigorously stirring for at least 24 h until the phase behavior could be properly established. The composition of the samples is expressed by surfactant weight percentage and molar fraction of cationic surfactant (mSer) in the mixture, as further defined in The catanionic samples were prepared by directly weighing the two respective surfactants into vials and adding Milli-QTo establish the phase behavior and detect colloidal aggregates (>1 µm), VELM imaging of the samples was performed with an Olympus BX51  microscope using bright-field illumination with differential interference contrast (DIC). The images were acquired with an Olympus DP71 digital video camera and the micrographs were processed with the Olympus CellA software provided by the manufacturer. The microscope was also used in polarized light mode to capture any birefringent structures .Solutions containing colloidal aggregates (namely vesicles) were vitrified and imaged with a JEOLJSM 6301F high-resolution scanning electron microscope, equipped with a Gatan Alto 2500 preparation chamber. Each sample was placed in a copper sample-holder and vitrified by plunging it from room temperature into liquid-nitrogen slush. The vitrified sample was then transferred to the preparation chamber to be fractured, sublimated at −90 °C (for 120 s) and sputtered with an Au/Pd coating (for 40 s) to render it electrically conductive. Afterwards, it was transferred to the microscope for imaging in secondary electron (SE) mode.ζ, (by ELS) were performed with a Zetasizer Nano ZS from Malvern Instruments. A 4 mW He-Ne laser (operating at 633 nm) was used with a fixed scattering angle of 173° (for particle size) or 17° . For both DLS and zeta potential measurements, 5 repeats per sample (minimum) were performed. The autocorrelation function was analyzed using the Malvern Dispersion Technology Software (DTS), with multiple narrow mode (high resolution) data processing. Given the multimodal or polydisperse unimodal character of the observed distributions, a non-negative least squares (NNLS) algorithm was employed [μ, was measured using electrophoresis and laser Doppler velocimetry techniques, and ζ was calculated from μ using the Henry equation, with a dielectric constant of 78.5, a medium viscosity of 0.89 cP, and a f(κa) function value of 1.5 (Smoluchowsky approximation) [Measurements of particle size (by DLS) and zeta potential, employed ,62. The imation) ,62. ® using a Whilelmy Pt/Ir plate. The temperature was kept constant using a Julabo® thermostated circulating bath, at 25.0 ± 0.1 °C. The surface tension of either neat surfactant or catanionic solutions was measured with a DCAT 11 tensiometer from Dataphysics− (aq) + H2O (l) → RCOOH (s) + OH− (aq). For this reason, the pH had to be adjusted to 12 with KOH to remove all the precipitated acid. Additionally, the Krafft temperature of the cationic surfactant 14Ser was measured as ≈26–27 °C. However, when investigating the 12Ser/8-8Ser and 14Ser/10-10Ser mixtures, it was realized that both originated extensive solution regions without any previous pH adjustment and at room temperature. Thus, the phase studies were performed at 25 °C and non-adjusted pH, employing direct ocular observations, VELM and cryo-SEM, and looking mainly for the formation of discrete colloidal aggregates  in solution. The samples were prepared at a constant 0.5 wt% total surfactant concentration—i.e., 99.5 wt% water, meaning these were highly diluted samples—and with varying proportions between the surfactants. The latter are expressed as the molar fraction of cationic surfactant, defined as xmSer = nmSer/(nmSer + nn-nSer), where n is the number of moles of surfactant. Alternatively, we also use the molar ratio between surfactants, defined as R+/− = nmSer/nn-nSer.The phase behavior and aggregation properties of the two catanionic mixtures were initially investigated and the results are shown in xmSer ≤ 0.40, i.e., for excess double-chained anionic surfactant, phase separation occurs in the form of a crystalline solid precipitating out of solution. This solid is most likely the 1:1 stoichiometric complex (catanionic surfactant), since when these samples were heated up to 80 °C, it remained totally insoluble; however, no further investigations on this issue were performed. Significantly, for 0.40 < xmSer ≤ 0.90, a rather wide composition range, solutions with a bluish hue  form, indicating the presence of large aggregates. For xmSer > 0.90, where there is a high excess of cationic surfactant, colorless and weakly scattering solutions appear . Secondly, microscopy imaging of the intermediate bluish solutions of both systems clearly show that the large aggregates are vesicles. Representative micrographs A–C (VELM) and A′–C′ (cryo-SEM) are provided in Both systems show common features in terms of macroscopic phase behavior, as depicted in Having established the presence of catanionic vesicles in the two mixtures, it was important to answer several other questions: (i) what are the vesicle size distribution, vesicle charge, and the pH of these solutions?; (ii) are vesicles the only aggregates present in the bluish solution region?; (iii) knowing a priori that the neat cationic surfactants self-assemble into micelles, how does the vesicle-to-micelle transition progress as cationic surfactant is added?; (iv) what is the critical aggregation concentration of vesicle-containing solutions? In order to address these questions, DLS and surface tension measurements were carried out, and the results and insight withdrawn are presented in the next two sections.DH> (± standard deviation), is plotted vs. molar fraction of cationic surfactant. Below each plot, representative intensity-weighted size distributions from DLS are provided to illustrate how the upper plots were constructed and to make comparisons. The results of DLS measurements, performed in the solution regions of the two catanionic systems, are shown in x12Ser < 0.70. It is only between ≈ 0.70 and ≈ 0.88 that they are seen coexisting with micelles, detected in the DLS as aggregates of ca. 4–6 nm. Moreover, and interestingly, within the vesicle region, the population is bimodal for x12Ser ≈ 0.40–0.60  but become unimodal, with <DH> ≈ 250 nm, in the narrow region of 0.60 < x12Ser < 0.70; these distributions remain stable for at least 6 months. Additionally, and significantly, the vesicles of the 0.70–0.88 coexistence region remain constant in size at ≈ 250 nm. This strongly suggests that they have a particular average composition responsible for their stability, corresponding to x12Ser ≈ 0.65 or R+/− ≈ 2:1. We will come back to this point in the discussion While the aggregation behavior is similar between the two systems up to a molar fraction of cationic surfactant of ≈ 0.40, there are a few key differences in the region 0.40–1. Analyzing first the 12Ser/8-8Ser system, vesicles are the single form of assembly in the approximate range 0.40 < x14Ser. Contrary to the previous system, this supports the notion that the vesicle composition changes with the sample composition. Thirdly, the coexisting micelles have an average size ranging between 11 and 19 nm, more than twice as large as that of the 12Ser/8-8Ser mixed micelles. This result is qualitatively unsurprising, given that the chain lengths involved in the 14Ser/10-10Ser are larger, and thus should lead to larger cross-sectional lengths, irrespective of the exact micelle shape. Overall, the picture that emerges is that the 14Ser/10-10Ser mixture is more polymorphic and polydisperse than the 12Ser/8-8Ser, given that for the former the vesicle/micelle region is wider, the vesicles change with mixing ratio and single vesicles are only found as bimodal populations. The deeper significance of these results will be further discussed in In the 14Ser/10-10Ser system, three important differences arise. First, vesicles as single aggregates are found only for an extremely narrow composition region (≈0.40–0.55) and as a bimodal population (≈80 and 300 nm). Secondly, though the vesicles coexist with the micelles for the entire 0.60–0.90 range, the vesicle average size is not constant, instead decreasing with increasing ζ and pH are essentially similar between the two mixtures, but some subtle differences also surface. The ζ values in these systems depend on several varying factors, in an intricate way: system composition, size and shape of the aggregates, pH and ionic strength. Regarding the pH, it is affected by the acid/base equilibrium of the n-nSer surfactants, by the intrinsic acidic pH of the cationic surfactants (as shown by the pH at xmSer = 1), and presumably also by the aggregation state of the system. A detailed and unequivocal analysis of the results is virtually impossible, yet some trends can be qualitatively withdrawn and rationalized.Analyzing now the zeta potential and pH plots of ζ ≈ −90 mV; they are also alkaline, with a pH ≈ 11–12  ζ is roughly constant and of about +32 mV, while for the 14Ser/10-10Ser vesicles it is larger, +47 mV, increasing smoothly in the vesicle/micelle region up to +65 mV at x14Ser = 0.80. This increase is not surprising, and essentially reflects the increase in cationic surfactant content in the systems. Yet, for the 12Ser/8-8Ser system, ζ drops to about +22–23 mV in the x12Ser = 0.70–0.88 region, even though the system’s cationic content is still increasing; this decrease seems to reflect the deep morphological change in the system on going from vesicles to coexisting vesicles and micelles. A fall-out in ζ also takes place (even more sharply) in the 14Ser/10-10Ser, but at x14Ser ≥ 0.90, where basically only highly cationic micelles should be present. Hence, we can conclude that aggregate size, shape, and coexistence strongly affect ζ in these systems.The neat 8-8Ser and 10-10Ser vesicles  to acidic (≈5) in both systems, with neutral pH occurring for roughly R+/− = 1, xmSer = 0.50) vesicles in both systems are, contrary to expectation, strongly cationic, specifically with ζ = +31 mV for the 12Ser/8-8Ser and ζ = +49 mV for the 14Ser/10-10Ser vesicles, whereas one might have expected values near 0 mV. It can be seen from −/RCOOH equilibrium, with a consequent contribution to an increase in ζ. We cannot exclude that there could be a differential composition between the outer and inner monolayer, a hypothesis proposed previously for the stabilization of catanionic vesicles [A striking feature is that even the equimolar (vesicles . An innecac). Surface tension measurements were performed for the neat surfactants and for two compositions of the mixtures, xmSer = 0.50 (vesicles) and 0.80 (vesicle/micelle coexistence), as shown in cac, the surface tension at that point (cacγ), as well as the minimum surface area per molecule (sa), calculated through Equation (1) (Gibbs adsorption isotherm) and Equation (2), were determined. For the mixtures, it is only possible to determine cac and cacγ. The Gibbs isotherm equation applied to a surfactant solution may be expressed as:sΓ is the surface excess of surfactant (for zero surface excess of the solvent), R is the gas constant, T is the temperature, sn is the number of adsorbed species obtained from the surfactant at the interface (Gibbs pre-factor) and dγ/dln(sc/oc) is the local slope of the γ vs. ln(sc/oc) curve . The minimum surface area per surfactant molecule is calculated from:AN is Avogadro’s constant and s,maxΓ is the maximum surface excess calculated for the biggest slope of the γ vs. ln(ic/oc) curve prior to the cac inflection point.An important aspect to consider when vesicles are used as membrane models or molecular nanocarriers is their critical aggregation concentration (ns in Equation (1) is equal to 2 (charged amphiphilic ion + respective counterion). However, for 8-8Ser and 10-10ser, due to pH adjustment with KOH, the large excess of small ions should partially screen the interactions at the interface, and hence make ns closer to 1 than to 2 [sa values for 8-8Ser and 10-10ser in sn = 1 and 2. We may reasonably infer that these sa values are likely closer to 1.2 and 1.0 nm2, respectively (2), since the double-chained surfactants have bulkier hydrophobic portions, hence requiring more critical area in the adsorbed monolayer. Consistently, the surface tension at cac, cacγ, is significantly lower for 8-8Ser (23.6 mN·m−1) and 10-10Ser (23.2 mN·m−1), than for 12Ser (30.2 mN·m−1) and 14Ser (32.9 mN·m−1). This reflects the higher tendency of the double-chained unimers to be surface-adsorbed than the single-chained ones.For ionic surfactants in neat water, typically the number of adsorbed species han to 2 ,58. Haviectively . This iscac. 8-8Ser and 10Ser-Ser possess cac values of 0.32 and 0.10 mmol.dm−3, respectively . 12Ser and 14Ser, which unequivocally aggregate into micelles, have cmc values of 3.0 and 1.2 mmol.dm−3, respectively , with a very short “plateau”. This type of profile, with two breaking points (i.p. + cac) in the surface tension curves, has been reported before for some catanionic mixtures [cac, with the consequence that at the cac there is coexistence of two types of aggregates. In both mixtures, for xmSer = 0.80, there are indeed coexisting micelles and vesicles; for xmSer = 0.50, two vesicle populations coexist . In both cases, the curve profile would thus be qualitatively justified and consistent with the DLS data. Despite its relevance, we did not investigate this issue any further.Looking at the surface tension curves of the mixtures, there is a clear mixtures ,64 and omixtures . The i.pcac of the mixtures, compared to the cac or cmc of the individual surfactants, several interesting facts arise. For the 12Ser/8-8Ser system, the cac values of the 0.50 and 0.80 samples are similar between them, about 40–50% of the cmc of 12Ser and about 5–6 times the cac of 8-8Ser. This indicates that the presence of the double-chained surfactant does not have a particularly great effect in decreasing the cac of this mixture. In other words, the 12Ser/8-8Ser catanionic vesicles form for concentrations of the order of 1.0 mmol·dm−3, a value sizably bigger than usual for vesicle-forming systems (≈10−6–10−3 mmol·dm−3). Regarding the cacγ values of 0.50 and 0.80, they are in between those of 12Ser and 8-8Ser, but for the equimolar composition (≈25 mN·m−1) very close to that of 8-8Ser (≈24 mN·m−1), suggesting that here the gas–liquid interface is enriched in the double-chained surfactant.If we now analyze the cac (0.20 mmol·dm−3) markedly smaller than that of the 0.80 (0.49 mmol·dm−3) and closer to the cac of 10-10Ser (0.10 mmol·dm−3). This means that, contrary to the previous mixture, the double-chained surfactant here induces a large decrease in cac. Moreover, the 14Ser/10-10Ser vesicles form at concentrations much smaller than the 12Ser/8-8Ser vesicles (0.2/1.2 ≈ 6 times), a feature that is interesting for drug delivery applications . The cacγ values of x14Ser = 0.50 and 0.80 are virtually identical to those of 14Ser, and this suggests that the gas–liquid interface is enriched in the single-chained surfactant. Overall, and given that the nature of the headgroups is exactly the same in both mixtures, as well as the asymmetry in chain length (n − m), we can conclude that the total chain length (n + 2m) has a marked effect on the aggregation and interfacial properties of these mixtures.Turning now to the 14Ser/10-10Ser system, the features are fairly different. The 0.50 sample has a sP) model [sP is defined as the ratio hcv/(o lca), where hcv and cl are the volume and critical length of the hydrophobic portion of the molecule and oa is the critical headgroup area at the aggregate interface. The surfactant’s molecular shape and respective Ps dictate the type of favored aggregate: for an inverted cone and sP = 0.33, spherical micelles; for an inverted truncated cone and sP ≈ 0.33–0.5, cylindrical micelles; for a wedge-shaped amphiphile and sP ≈ 0.5–1, bilayers disks and vesicles; for a cylinder-like shape and sP ≈ 1, planar bilayers; and for a truncated cone with sP > 1, reverse structures. The double-chained surfactants 8-8Ser and 10-10Ser have a sP ≈ 1 and hence form vesicles/bilayers, as we confirmed (by VELM), while the single-chained 12Ser and 14Ser, with sP ≈ 0.33 form small micelles, as detected by DLS. We can interpret self-assembly in catanionic mixtures resorting, as a first approach, to the surfactant packing parameter (s) model . Ps is dxmSer), both electrostatic headgroup interactions and chain packing effects take place. Initially, when 0 < xmSer ≤ 0.40, the electrostatic association between the two surfactants , the excess positive charge and curvature effects (dictated by the intrinsic Ps ≈ 0.3 of mSer) prevail, and therefore bilayers are no longer the single aggregation state. The system responds in the form of coexisting vesicles and micelles . When xmSer > 0.90, only mixed micelles are stable and bilayers disappear from the system, as expected.When the cationic mSer surfactant is gradually added to each mixture (increase in  release ) dominatIn catanionic mixtures, the transition between micelles and vesicles usually occurs via two routes . One rouDH> = 200 nm, and vesicle/micelle coexistence involves large wormlike micelles. The 12Ser/12-12Ser system is dominated by precipitation at room temperature and mixed polydisperse vesicles only form at 60 °C. The 12Ser/8-8Ser and 14Ser/10-10Ser have very different features from both, being intermediate between the highly asymmetric case (sharing with it formation of vesicles and micelle/vesicle coexistence at room temperature) and the symmetric case (sharing solid–liquid phase separation).We can also directly compare these systems to the 16Ser/8-8Ser  and 12Ser/12-12Ser (symmetric) systems previously investigated by us , since tκ) is high. This is the case of charged and relatively rigid catanionic bilayers. It implies that only narrow mixing ratio ranges (slightly non-equimolar) are optimal for vesicle stability and any deviations dictate an unfavorable bending energy. CTAB/sodium perfluorooctanoate [κ (≈ few kBT), i.e., relatively soft flexible catanionic bilayers, an entropic stabilization takes place leading to big and polydisperse vesicles, and wider surfactant mixing ratios for vesiculation. Here, chain length asymmetry and packing effects of dissimilar hydrocarbon chains contribute to decrease κ and promote stabilization. This is assumed to be the case of CTAB/SOS [R+/− = 2:1 (DLS data). These features are more akin to those of a curvature stabilization mechanism, (A), than to an entropic one, (B). In contrast, the more polydisperse 14Ser/10-10Ser vesicles and the variation in vesicle size depending on sample composition seem to underpin an entropic stabilization mechanism.Several models have been proposed to explain vesicles as equilibrium aggregates in catanionic mixtures  rather than as a metastable dispersed form of a lamellar liquid crystal ,69,70. Wctanoate  and 12Sectanoate  vesiclesCTAB/SOS  and, sigCTAB/SOS  vesiclesR+/− ≈ 2:1. They also have <DH> ≈ 250 nm, ζ = +35–40 mV and pH ≈ 7–8, forming at a total surfactant concentration ≥ 1 mmol·dm−3. Overall, they show interesting characteristics for biomolecular delivery. The 14Ser/10-10Ser vesicles instead form for a narrower compositional region and appear only as bimodal, polydisperse populations (80 and 300 nm), ζ = +45–50 mV and pH ≈ 9–10, forming at smaller concentrations (≥0.2 mmol·dm−3).Herein, we showed that two mixed cationic/anionic surfactant systems based only on serine-derived surfactants, 12Ser/8-8Ser and 14Ser/10-10Ser, are able to form stable vesicles at room temperature, with no previous pH adjustment, under some specific mixture compositions . One first general conclusion is that these serine-based surfactants provide a source for preparing model, robust biomimetic vesicles than can be further explored for bio-related applications, namely for drug and (given their positive charge) gene delivery. These two mixtures share an identical surfactant chain length asymmetry (four carbon atoms), but different total alkyl chain size (28 versus 34 carbon atoms). A second general conclusion is that even if we retain chain length asymmetry, a relatively small change in total chain length has meaningful effects on the vesicle features. The 12Ser/8-8Ser vesicles exist either as bimodal or unimodal populations, in a wider compositional range. However, the unimodal vesicles seem to have a specific average composition of


This electrochemical nanosensor is suitable for point-of-care detection of PT in a wide dynamic range of 3 × 10−11–11 × 10−6 M with a lower detection limit of 10.9 pM. The performance of the nanosensor was validated by adding PT to natural samples and comparing the data via absorption spectroscopy. PT detection results encourage the design of easy-to-use nanosensor-based analytical tools for rapidly monitoring other environmental samples.Organophosphate-based pesticides ) have toxic effects on human health through their residues. Therefore, cost-effective and rapid detection strategies need to be developed to ensure the consuming food is free of any organophosphate-residue. This work proposed the fabrication of a robust, nonenzymatic electrochemical-sensing electrode modified with electrochemically reduced graphene oxide (ERGO) to detect PT residues in environmental samples  as well as in vegetables and cereals. The ERGO sensor shows a significantly affected electrocatalytic reduction peak at −0.58 V (vs Ag/AgCl) for rapid quantification of PT due to the amplified electroactive surface area of the modified electrode. At optimized experimental conditions, square-wave voltammetric analysis exhibits higher sensitivity (50.5 μA·μM Crop production is constantly increasing to fulfil the demands of the growing population. The protection of crops against insects is a big challenge for our society. Pesticides have indiscriminately been used in all sectors of agriculture. Among the various pesticides available for the aforementioned purpose, organophosphates (OPs) are commonly employed in agriculture, households, gardens, and veterinary practices. This practice also jeopardizes food safety in all stages of the food supply chain, even after pesticide use. Due to its high nondegradability, pesticides can stay more often on the surface of fruits and vegetables; sometimes, it can also penetrate into the peel of vegetables and fruits . OrganopThe high-throughput analytical methods such as chromatographic  and spectroscopic  techniques are time-consuming, laborious, costy, require specific and sophisticated instruments and trained personnel, and most often are not portable to enable on-site detection –6. Elect2, and CuO–TiO2 hybrid nanocomposites were proposed to detect methyl parathion 3− and [Fe(CN)6]4− with 0.1 M KCl within the frequency range from 1 MHz to 0.01 Hz, amplitude of 10 mV, at a fixed potential of 0.28 V.Voltammetric studies were carried out using an IVIUMStat electrochemical analyzer , which was connected by a three-electrode system, including a modified and/or unmodified GCE as the working electrode, a saturated Ag/AgCl as the reference electrode (RE), and a platinum wire as the counter electrode (CE). The electrochemical impedance spectroscopy (EIS) study of the modified electrodes was carried out in 5 mM of  + 12.018 (R² = 0.9871) for the range of 0.1–11 μM, Ip (μA) = 0.2916 [PT] + 3.7526 (R² = 0.9936) for 3–15 nM, Ip (μA) = 19.176 [PT] + 4.2723 (R² = 0.9367) for 0.03–0.15 nM. The corresponding sensitivity was found to be 50.5 μA·μM−1·cm−2 with a wide linear range for quantification of PT. The limit of detection (LOD = [(3 * standard deviation of blank)/slope of the lowest range of linear curve ] and limit of quantification (LOQ = [(10 * SD of blank)/slope]) were calculated as 10.9 pM and 36.5 pM, respectively, from the lower calibration equation [equation ,41.+, K+, Fe2+, Fe3+, Cd2+, Ni2+, Mg2+, Mn2+, NH4+, Cl−, NO3−, CO32−, SO4−, CH3COO-), nitroaromatic compounds , and other pesticides, such as acephate, chlorpyrifos, dicofol, lindane. As shown in The selectivity of the proposed ERGO/GCE modified nanosensor  was inveReproducibility, repeatability, and stability are essential parameters for practical applications of electrochemical nanosensors. Inter-assay measurements of 10 μM PT using five independent ERGO/GCE were performed, and a 3.4% relative standard deviation (RSD) was obtained for five replicate scans, indicating good reproducibility of the proposed nanosensor. Similarly, a single modified electrode exhibits good repeatability with an RSD of 1.81% for five repeated measurements performed in PBS  containing 10 μM PT.The analytical performance of the ERGO/GCE, such as detection limit and linear range, are compared with previously reported modified electrodes for the detection of PT  16–183618–44. The 18To determine the storage stability, the electrocatalytic response of 10 μM PT was monitored in seven-day intervals for the first two months, and it retained about 96.17 ± 0.2% of its initial response. It was shown a consistent response to PT sensing during two months of storage. After that, the response was measured in intervals of 10 days, and 90.53 ± 0.3% of the initial response was retained after six months. This indicates good stability of the modified electrode at room temperature (−11–11 × 10−6 M), and fast response time. The proposed ERGO/GCE nanosensor exhibits excellent electrocatalytic activity, long-term storage stability, reproducibility, repeatability, low-cost fabrication, and strong anti-interference ability to quantify PT residues in real samples. The low RSD value (0.998–1.62%) and relative error (1.9–3.9%) obtained from UV data confirmed the accuracy of this method, showing that the electrochemical nanosensor has good reliability for PT detection in real samples. It can be concluded that the feasible nonenzymatic electrochemical nanosensor could be a good alternative for on-site monitoring of PT usage in agricultural fields. The robust and straightforward electrochemical-sensing platform could also be a promising path for selective and sensitive analysis of other pesticides and environmental pollutants based on electrocatalytic activity.A newly developed inexpensive and environmentally friendly techique, using an interference-free nonenzymatic approach was developed to fabricate a nonenzymatic electrochemical nanosensor based on ERGO for rapid detection of PT. The electrochemical parameters were optimized to achieve the highest performance of the ERGO-modified electrode, and the structure was characterized by Raman, XRD, XPS, TEM, FESEM, and EIS techniques. Square-wave voltammetry was performed to achieve excellent nanosensor performance, such as higher sensitivity, low detection limit (10.9 pM), linear response range (3 × 103Fe(CN)6 solution with 1 M KCl. Figure S2: (A) Storage stability of the proposed sensing matrix (ERGO/GCE), (B) SWV of PT  added in groundwater. Figure S3: (A) Absorption spectra of parathion and (B) corresponding calibration plot.Table S1: Chemical sample table indicating corresponding CAS, supplier and other details. Table S2: Experimental sample table for composition analysis using binding energies of GO and RGO by XPS. Table S3: Experimental sample table for the modified glassy carbon electrode electrochemical characteristics. Figure S1: CV of ERGO/GCE at different scan rates (10–300 mV/s) in 1.0 mM KFile 1Additional figures and tables.