Aspalathin is a rooibos flavonoid with established blood glucose lowering properties, however, its efficacy to moderate complications associated with hepatic insulin resistance is unknown. To study such effects, C3A liver cells exposed to palmitate were used as a model of hepatic insulin resistance. These hepatocytes displayed impaired substrate metabolism, including reduced glucose transport and free fatty acid uptake. These defects included impaired insulin signaling, evident through reduced phosphatidylinositol-4,5-bisphosphate 3-kinase/ protein kinase B (PI3K/AKT) protein expression, and mitochondrial dysfunction, depicted by a lower mitochondrial respiration rate. Aspalathin was able to ameliorate these defects by correcting altered substrate metabolism, improving insulin signaling and mitochondrial bioenergetics. Activation of 5ʹ-adenosine monophosphate-activated protein kinase (AMPK) may be a plausible mechanism by which aspalathin increases hepatic energy expenditure. Overall, these results encourage further studies assessing the potential use of aspalathin as a nutraceutical to improve hepatocellular energy expenditure, and reverse metabolic disease-associated complications. Obesity and dysglycemia, the major characteristic features of the metabolic syndrome, have become prevalent in the general population and are associated with a rapid rise in morbidity and mortality , 2. InsuDespite the acknowledged importance of the PI3K/AKT pathway in insulin signaling and cell survival , completAspalathus linearis, a South African plant used to manufacture the widely consumed rooibos herbal tea, as nutraceuticals -D-glucose and 14C palmitate were obtained from American Radiolabeled Chemicals , while Bradford and RC DC protein assay kits, and Mini-PROTEAN TGX Stain-Free Precast Gels, nitrocellulose and PVDF membranes were from Bio-Rad Laboratories . Eagle's Minimum Essential Medium (EMEM), Dulbecco’s phosphate buffered saline , penicillin-streptomycin and MycoAlert mycoplasma detection and ViaLight plus ATP kits were from Lonza , while fetal calf serum (FCS) was obtained from Biochrom . Chemiluminescence (ECL) kit was from Amersham Bioscience . Primary antibodies raised against AKT (cat# 9272S), p-AKT (Ser473) (cat# 9271S), AMPK (cat# 2532), p-AMPK (Thr172) (cat# 2535S), carnitine palmitoyltransferase 1A (CPT1) (D3B3) (cat# 12252S), PI3K (cat# 4257S), p-PI3K (p85) (cat# 4228S) were from Cell Signalling Technology , while glucose transporter (GLUT) 2 (cat# ab54460) was from Abcam . The reference control, β-actin, and secondary antibodies, goatanti-mouse and goatanti-rabbit IgG–horseradish peroxidase, were purchased from Santa Cruz Biotechnology . Seahorse XF96 microplate plates, Seahorse XF Assay media, Seahorse XF base media without phenol red and Seahorse XF-Cell Mito Stress, Glycolysis stress assay kits were from Agilent . All other cell culture reagents were obtained from Sigma-Aldrich Aspalathin , synthesized following an already published method by Han et al. , was sup3H]-D-glucose and 14C palmitate uptake experiments, 96-well microplates (11 000 cells/well) for ATP and MTT assays and 6-well plates (165 000 cells/well) for protein analysis. For Seahorse experiments, cells were seeded at 20 000 cells/ well in 96-well Seahorse microplates until confluent. C3A cells were confirmed to be free from mycoplasma before successive experiments, using the MycoAlert mycoplasma detection kit according to the manufacturer’s instructions. Cells were maintained by rinsing with DPBS and trypsinization by adding 2 ml trypsin/ versen to the cell culture, and incubation for 3–7 min until cells dislodged. Thereafter, cells were seeded in EMEM with essential amino acid and L-glutamine for 5-days and incubated under standard culture conditions at (37°C in 5% CO2 and humidified air), and media was changed every 48 hours. Cell passages between 5 and 20 were used for all experiments. Insulin resistance was induced by exposing C3A liver cells to 0.75 mM palmitate dissolved in EMEM containing 5.5. mM glucose and 2% bovine serum albumin for 16 h, as previously described -D-glucose and 14C palmitate uptake assays were performed separately, as previously described [3H]-D-glucose or 1 μCi/mL 14C palmitate in medium containing aspalathin. Insulin (1 μM), the positive control, was added during the last 15 min in each experiment. Thereafter, cells were lysed with 0.1 M NaOH before 2-deoxy-[3H]-D-glucose and 14C palmitate activity in the lysate was assessed by liquid scintillation .2-Deoxy-[escribed , 26. BriIntracellular ATP was measured using the ViaLight plus ATP kit, following manufacturer’s recommendations, whereas the MTT -2,5-diphenyltetrazolium bromide) assay was done according to a previously described method . For the2 for 60-min to equilibrate temperature and pH prior to OCR. The XF96 plate was then transferred to a temperature-controlled (37°C) Seahorse XF96 extracellular flux analyzer where it was subjected to a 10 min equilibration period at three assay cycles, each including a 1 min mix, 3 min wait and 3 min measure period cycle. Following this, oligomycin (1 μM) was injected in port A to inhibit ATP synthase. After 3 assay cycles, 75 μM carbonyl cyanide 4 trifluoromethoxy-phenylhydrazone (FCCP) was added in port B to measure maximal respiration. A mixture of Complex I inhibitor (rotenone) and complex III inhibitor (antimycin) was injected in port C to shut down mitochondrial respiration and enable the calculation of non-mitochondrial respiration. At the end of the incubation period, the plates were used to assess protein concentration in each well by Bradford protein assay. OCR (pmol/min) was normalized based on protein content. OCR and ECAR were reported as absolute rates (pmoles/min for OCR and mpH/min for ECAR).Oxygen consumption rate (OCR) and extracellular acidification rates (ECAR) were measured with the Mito Stress assay kit, using the XF96 Extracellular Flux analyzer from Agilent. Briefly, C3A liver cells were plated into cell culture XF96 microplate plates at 20,000/well for 48 h before treated as previously described in the cell culture and treatment section. Following treatment, cells were washed twice with 180 μL of pre-warmed assay medium , thereafter, cells were incubated in 180 μL XF Assay Medium at 37°C without CO2 incubator at 37°C for 60 min before the assay. For measurement of acute glycolysis, the compound of interest  or buffer was injected in port A followed by injections of glucose (10 mM), oligomycin (1 μM) and 2-DG (100 mM) into ports B, C and D, respectively. The instrument was calibrated, and the assay was performed using a glycolytic stress test assay protocol as recommended by the manufacturer (Agilent Technologies). ECAR was measured under basal conditions followed by the sequential addition of aspalathin, 10 mM glucose, 0.5 μM oligomycin, and 100 mM 2-DG (ASP> Glu> Oli> 2-DG). Thereafter non-glycolytic acidification, glycolysis, glycolytic capacity and glycolytic reserve were calculated using Wave desktop (Agilent Technologies).After a 3 h incubation with relevant treatment, the medium was changed to assay medium (XF base medium DMEM without phenol red supplemented with 2 mM glutamine), and cells were incubated in a non-COA standardized protocol was used for Western blot analysis . Brieflyt-test. A p value of < 0.05 was considered statistically significant. For cellular respiration experiments, the XF Mito Stress test report generator and the XF glycolysis stress test report generator automatically calculated the respective parameters from Wave data that have been exported to Excel. Respiration and acidification rates are presented as the mean ± SEM of 2 independent experiments in all experiments performed, with 4 to 8 replicate wells in the Seahorse XF96 analyzer. For the experiment of the effect of energy substrates on mitochondrial respiration, the significance level was determined by performing ANOVA on the complete data set with Tukey׳s post-hoc testing. The results were considered significant at p < 0.05.Data are presented as the mean ± SEM of three independent experiments. GraphPad Prism 5 software  was used for all statistical analyses. Comparisons between treatment groups were performed by one-way analysis of variance (ANOVA), followed by a Tukey post hoc test and Student Glucose uptake assay was used to determine the most active concentration of aspalathin in C3A liver cells. Results showed that together with both insulin and metformin, all tested concentrations of aspalathin (0.0001–100 μM) improved glucose uptake in cells . HoweverLike in many cells, FFAs and glucose are the major substrates utilized by hepatocytes to generate energy. Impaired hepatic substrate metabolism was confirmed by a significant reduction in FFA and glucose uptake after palmitate exposure (p < 0.001) . A similThe PI3K/AKT pathway represents one of the most investigated mechanisms in connection with pathological complications of insulin resistance. Although no changes were observed under basal conditions, exposure of hepatocytes to elevated palmitate concentrations reduced phosphorylation of AKT (p < 0.001) in insulin-stimulated cells when compared to the normal control . No signModulation of AMPK, together with CPT1 is increasingly targeted for the regulation of energy metabolism, especially FFA oxidation. Exposure of hepatocytes to elevated palmitate concentrations significantly reduced protein expression of CPT1 (p < 0.001), and slightly enhanced AMPK (p < 0.001) . HoweverThe MTT and ATP monitoring assays are widely used to measure the metabolic activity of viable cells. While no effect was observed after cells were exposed to high palmitate concentrations with MTT assay, ATP production was significantly reduced (p < 0.01) when compared to the control . HoweverMitochondrial respiration was measured through chronological injections of oligomycin, FCCP, antimycin A and rotenone to assess basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration . CombineThe rate of glucose conversion into pyruvate (glycolysis) was determined after injecting with or without aspalathin in port A, . Albeit The rapid rise in deaths due to noncommunicable diseases warrants further investigation into novel drugs that can act broadly to alter energy metabolism or influence pathways that contribute to metabolic disease-associated complications. Hepatocytes play a major role in regulating energy metabolism, in addition to their established function in the detoxification of metabolites and synthesis of proteins. Abnormal hepatic energy metabolism has been associated with the development of various metabolic complications, including NAFLD, insulin resistance, and diabetes mellitus , 14, 35.Exposing C3A cells to high palmitate concentrations led to a number of metabolic and cellular signaling abnormalities that are related to the development of insulin resistance. Such abnormalities included altered substrate metabolism, mainly characterized by suppressed glucose and FFA uptake, as well as reduced GLUT2 expression, occurring concomitant to altered insulin signaling. Reduced phosphorylation of both PI3K and AKT could explain impaired insulin signaling and development of insulin resistance, as previously demonstrated , 13, 31.Tripterygium wilfordii plant, could ameliorate hepatic insulin resistance by improving insulin signaling and mitochondrial function [in vitro. This result is consistent with our recent findings [in vitro, can be essential tools in establishing the involvement of PI3K/AKT or AMPK in the therapeutic effect of aspalathin.The major findings in this paper suggest that aspalathin can provide a number of beneficial effects that are essential in ameliorating hepatic-induced insulin resistance. Such benefits include tight regulation of hepatic substrate metabolism, which could have led to improved insulin signaling and mitochondrial bioenergetics. As reported by our group and others in different tissue types , 28, 31,function . Similarfunction . Whereasfunction . Thus, wfunction –46. Howefindings , showingin vivo model of insulin resistance. Such information would not only improve our current understanding on the mechanisms involved in metabolic disease associated liver dysfunction, but would also highlight the potential use of aspalathin as a nutraceutical to protect against metabolic disease-related complications.This study showed that aspalathin can target the liver cells to regulate hepatic cellular metabolism and increase energy expenditure likely by modulating PI3K/AKT and AMPK signaling pathways. However, before proposals presented in this study are accepted, several issues need to be resolved first. This includes confirming these results in an established S1 DatasetOriginal Western blot picture of glucose transporter (GLUT) 2, experiment 1(A), experiment 2(B), experiment 3 (C) and representative cropped blot (D). C3A liver cells were cultured in EMEM supplemented with 8 mM glucose with or without 0.75 mM palmitate for 16 h, and then treated with aspalathin 10 μM for 3 h. Insulin (1 μM) was added during the last 15 min and used as a positive control. Cells were lysed and subjected to Western blot analyses. All 3 independent experiments were analysed and one representative blot was cropped to be included in the article. Results are from three independent experiments. Please note: G-represents “normal control”, GI is “normal control+ insulin”, P is “palmitate control”, PI is “palmitate+insulin”, GRE is “green rooibos extract”, GRE+I is “green rooibos extract + insulin”, ASP is “aspalathin”, and ASP+I is “aspalathin+ insulin”.(DOCX)Click here for additional data file.S2 DatasetOriginal Western blot picture of phosphor-protein kinase B (pAKT) and total-protein kinase B (tAKT), experiment 1 , experiment 2 , experiment 3  and cropped blot (G). C3A liver cells were cultured in EMEM supplemented with 8 mM glucose with or without 0.75 mM palmitate  for 16 h, thereafter treated with aspalathin 10 μM for 3 h. Insulin (1 μM) was added during the last 15 min and used as a positive control. Cells were lysed and subjected to Western blot analyses. After probing with pAKT blots were stripped and probed with tAKT. The % of pAKT/tAKT, was used to estimate the level of AKT (Ser 473). All 3 independent experiments were analysed and one representative blot was cropped to be included in the article. Results are from three independent experiments. Please note: G-represents “normal control”, GI is “normal control+ insulin”, P is “palmitate control”, PI is “palmitate+insulin”, GRE is “green rooibos extract”, GRE+I is “green rooibos extract + insulin”, ASP is “aspalathin”, and ASP+I is “aspalathin+ insulin”.(DOCX)Click here for additional data file.S3 DatasetOriginal Western blot picture for phosphor-phosphoinositide 3-kinase (pPI3K) and total-phosphoinositide 3-kinase (tPI3K), experiment 1 , experiment 2 , experiment 3  and cropped blot (G). C3A liver cells were cultured in EMEM supplemented with 8 mM glucose with or without 0.75 mM palmitate  for 16 h, thereafter treated with aspalathin 10 μM for 3 h. Insulin (1 μM) was added during the last 15 min and used as a positive control. Cells were lysed and subjected to Western blot analyses. After probing with pPI3K blots were stripped and probed with tPI3K. The % of pPI3K/tPI3K, was used to estimate the level of PI3K (p85). All 3 independent experiments were analysed and one representative blot was cropped to be included in the article. Results are from three independent experiments. Please note: G-represents “normal control”, GI is “normal control+ insulin”, P is “palmitate control”, PI is “palmitate+insulin”, GRE is “green rooibos extract”, GRE+I is “green rooibos extract + insulin”, ASP is “aspalathin”, and ASP+I is “aspalathin+ insulin”.(DOCX)Click here for additional data file.S4 DatasetOriginal Western blot picture of carnitine palmitoyltransferase 1 (CPT1), experiment 1(A), experiment 2(B), experiment 3 (C) and cropped blot (D). C3A liver cells were cultured in EMEM supplemented with 8 mM glucose with or without 0.75 mM palmitate  for 16 h, and then treated with aspalathin 10 μM for 3 h. Insulin (1 μM) was added during the last 15 min and used as a positive control. Cells were lysed and subjected to Western blot analyses. All 3 independent experiments were analysed and one representative blot was cropped to be included in the article. Results are from three independent experiments. Please note: G-represents “normal control”, GI is “normal control+ insulin”, P is “palmitate control”, PI is “palmitate+insulin”, GRE is “green rooibos extract”, GRE+I is “green rooibos extract + insulin”, ASP is “aspalathin”, and ASP+I is “aspalathin+ insulin”.(DOCX)Click here for additional data file.S5 DatasetOriginal Western blot picture of phosphor-AMP-activated protein kinase (pAMPK) and total-AMP-activated protein kinase (tAMPK), experiment 1 , experiment 2 , experiment 3  and cropped blot (G). C3A liver cells were cultured in EMEM supplemented with 8 mM glucose with or without 0.75 mM palmitate  for 16 h, thereafter treated with aspalathin 10 μM for 3 h. Insulin (1 μM) was added during the last 15 min and used as a positive control. Cells were lysed and subjected to Western blot analyses. After probing with pAMPK blots were stripped and probed with tAMPK. The % of pAMPK/tAMPK, was used to estimate the level of AMPK (Thr172). All 3 independent experiments were analysed and one representative blot was cropped to be included in the article. Results are from three independent experiments. Please note: G-represents “normal control”, GI is “normal control+ insulin”, P is “palmitate control”, PI is “palmitate+insulin”, GRE is “green rooibos extract”, GRE+I is “green rooibos extract + insulin”, ASP is “aspalathin”, and ASP+I is “aspalathin+ insulin”.(DOCX)Click here for additional data file.S6 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Recently, we showed that LTB4 production by the 5-lipoxygenase (5-LO) pathway regulates IL-1β and PGE2 release, reducing tissue damage in a model of sterile inflammation. Since IL-1β and PGE2 increase in serum of stressed patients and potentially trigger depression, we used an animal model of chronic unpredictable stress (CUS) to investigate the potential impact of LTB4 over depression-like symptoms. At basal conditions, 5-LO deficiency (Alox5−/−) reduces the preference for sucrose, while inducing a higher immobilization time on the tail suspension test when compared 129sv. Moreover, Alox5−/− mice present increased caspase-1 expression and elevated levels of IL-1β, IL-17 and PGE2 in the spleen, with increasing corticosterone levels in the frontal cortex but reducing systemic levels. Compared to 129sv mice, CUS induced higher levels of systemic, frontal cortex and hippocampal corticosterone, and also reduced sucrose preference, increased levels of splenic IL-1β, IL-17 and PGE2 and reduced levels of LTB4. Interestingly, CUS exposure did not alter the reduced sucrose preference shown by Alox5−/− mice but greatly enhanced splenic PGE2 production. Compared to Alox5−/− mice at basal conditions, CUS exposure also increased levels of systemic corticosterone, which remained lower than those of CUS-129sv animals. We also observed that treatment with LTB4 decreased caspase-1 expression and systemic levels of corticosterone in CUS-Alox5−/− mice but there was no significant impact on the reduced sucrose preference. Our results demonstrate that LTB4 controls the hypothalamic-pituitary-adrenal (HPA) axis by regulating levels of systemic corticosterone associated with the repression of caspase-1 expression and production of inflammatory mediators. One limitation of our study is that 129sv and Alox5−/− mice were not littermates, not sharing, therefore, the same intra-uterine and preweaning environment. Even so, taken together our results indicate that 5-LO activity is critical for the regulation of stress-induced symptoms, suggesting that the Alox5−/− mouse could be a natural model of corticosterone-independent reduced reward sensitivity.Depression is a mental illness with a complex and multifactorial etiology, which has been associated with stress and inflammation. Infections, autoimmune diseases, envenomation, and trauma induce an inflammatory response that is characterized by increasing levels of circulating cytokines  and lipid mediators [e.g., PGE Depression is a serious public health problem and one of the most common psychiatric disorders in Western countries. It may lead to suicide, a tragic fatality accounting for mortality of 800,000 people every year . DepressDepressed and chronically stressed individuals exhibit higher levels of circulating proinflammatory cytokines, chemokines, acute phase proteins, and soluble adhesion molecules or even cerebrospinal fluid Smith, . InteresRecently, Zhang et al.  reported+ as a cofactor  and corticosterone (in rodents), are anti-inflammatory hormones , C4 (LTC4), D4 (LTD4) and E4 , the enzyme 5-lipoxygenase (5-LO) catabolizes arachidonic acid into leukotrienes (LT) Bz et al.  have sho2 increases IL-1β production by a cyclic adenosine monophosphate (cAMP)-dependent mechanism in a model of sterile inflammation  and 129sv mice to chronic unpredictable stress (CUS), an experimental model that induces depression-like behaviors such as decreased sucrose preference. Compared to 129sv mice at basal conditions, Alox5−/− animals exhibited an intrinsically lower preference for sucrose and higher levels of IL-1β, IL-17, PGE2, adrenocorticotropic hormone (ACTH), and caspase-1 expression. In contrast, these mice present lower 11βHSD2 and GR expression, as well as reduced levels of corticosterone, 11-dehydrocorticosterone (11DHC), and circulating neutrophils. As expected, 129sv mice exposed to CUS displayed increased levels of splenic proinflammatory and stress-induced mediators, and signs of reduced sucrose preference, which could be associated with anhedonia  were age-matched in all procedures with their genetic background, the wild-type 129sv mouse strain  and 129sv mice were obtained from The Jackson Laboratory  and were bred in the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo . C57Bl/6 mice, obtained from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, were used as controls to validate our methodology used in the 129sv mice. Mice were kept in 12 h light/dark cycles, with ad libitum access to water and food, except for the period of exposure to stressors (described in “Chronic Unpredictable Stress and Sample Collection” section). All procedures were performed according to the ethical standards established by the National Council for Control of Animal Experimentation (CONCEA) and approved by the Ethics Committee on Animal Use (CEUA) of the Campus of Ribeirão Preto (Protocol number: 14.1.290.53.5).Male mice (7–8-weeks-old) were used for the experiments. Animals lacking the 5-LO gene  for 24 h; reversal of the light/dark cycle; 15 min forced swimming; 1 h restraint in a tube. The last stressor was always water and food deprivation for 20 h. The next day, animals underwent a sucrose preference test to evaluate depressive-like behavior  and the blood was immediately collected by cardiac puncture for the analysis of circulating leukocytes. All blood samples were collected at 12:00 pm and in the presence or absence of an anticoagulant and mice were weighed at day zero, and again at day fifteen. Next, the animals were euthanized by cervical dislocation. The spleen and adrenal glands, and the cerebellum, frontal cortex, and hippocampus were weighed after collection.sv and Alox5−/− mice were individually housed and deprived of food and water (the last stress stimulus) for 20 h. Next, a glass bottle with water and another with water containing 1% sucrose was placed in each cage. The bottles were initially weighed and consumption was monitored after 1 h. For this purpose, the glass bottles were weighed again to estimate the liquid ingestion. The sucrose preference was calculated as described previously  injected with LTB4  during the morning. Treatment initiated 1 day before CUS, followed by administration twice a day (12/12 h) during the 14 days of CUS exposure. LTB4 was dissolved in ethanol and diluted in aqueous solution containing 0.45% NaCl, 0.25% dextrose, and 0.01% bovine serum albumin (BSA) followed by filter sterilization. The same volume of ethanol was added to the same aqueous solution to be used as the vehicle control. The LTB4 dose was selected based on previous studies , and the remaining plasma was stored at −20°C. ACTH concentrations were determined with a chemiluminescent immunoassay (CLIA) technology  employing a Liaison Analyzer , following the manufacturer’s instructions. The sensitivity of the assay was <1,500 pg/mL.Blood samples were collected and immediately used for total and differential counts of circulating leukocytes as previously described  coupled with the liquid chromatography system Nexera . Data were processed using PeakView and MultiQuant software. For spleens, PGE2 and LTB4 levels were determined using a LC-MS/MS system Acquity UPLC®—Xevo TQ-S . Data were analyzed with MassLynx 4.0 and TargetLynx 4.0 softwares.Corticosterone and 11DHC from serum, frontal cortex, hippocampus, and cerebellum were quantified by mass spectrometry (MS) as previously described  according to the manufacturer’s instructions. The reading was taken at 405–420 wavelength and the results were expressed in ng/mL.Spleens were homogenized  and commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kits  were used for quantification of IL-17 and IL-1β levels, according to the manufacturer’s instructions. The sensitivity of the assay was <10 pg/mL.6 cells/mL and flow cytometry was carried out as previously described  staining , according to the manufacturer’s instructions. Briefly, cellular suspensions were washed twice with PBS, centrifuged at 400× g for 15 min, suspended in the kit buffer, and then incubated with annexin V in the absence of light for 15 min. PI was added to all samples immediately before data acquisition. A total of 10,000 events were acquired from all samples using the FACSCanto flow cytometer . The results were evaluated using the FACSDiva software and expressed as the percentage of cells in the initial stage of apoptosis, i.e., positive for annexin V and negative for PI . Total cell suspensions were adjusted to 1 × 10Hsd11b1, Hsd11b2, and Casp1 genes employing StepOne Plus . Actb was used as the endogenous control. The reactions were amplified under the following conditions: denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 2 min and 60°C for 20 s. The expression of the genes Hsd11b1 , Hsd11b2 , and Casp1  were normalized by housekeeping gene expression (Actb). The gene expression of 129sv mice was considered as the reference (129sv mice), being 2−ΔΔCT = 1. The primers were obtained commercially and are proprietary; thus, sequences are not available .Adrenal glands and spleens were collected, and total RNA was extracted using PureLink RNA Mini Kit , according to the manufacturer’s instructions. RNA concentration and purity were evaluated using a fluorimetric assay . Complementary DNA (cDNA) was obtained using the High Capacity cDNA Reverse Transcription kit , according to the manufacturer’s instructions. Total cDNA was amplified by real-time reverse transcription polymerase chain reaction (RT-qPCR) using TaqMan primers for the g for 10 min, the supernatant was used to measure GR expression. Equal amounts of protein (50 μg) from each sample were separated by polyacrylamide gel for electrophoresis (10% Tris-HCl) and subsequently transferred to a nitrocellulose membrane, as previously described . After homogenization and centrifugation at 400× sv and Alox5−/− mice, exposed or not to CUS. Briefly, all samples were homogenized  in KCl buffer  and centrifuged at 1,000 g for 10 min 4°C. Protein assay was performed on the supernatant using Coomassie blue staining, as described previously by Medeiros et al. , when appropriated. Post hoc comparisons were performed with the Newman-Keuls test. Differences were considered significant when p < 0.05.Data were analyzed using SPSS V. 20 . After checking for normality and homoscedasticity, the results were analyzed using Student’s sv. The former strain has been widely characterized in studies addressing CUS-induced depression-like behavior  = 42.06, p < 0.001), but corticosterone levels remained comparable between them  = 7.13, p < 0.02; sv compared to CUS-C57Bl/6  = 6.83, p = 0.017; sv mice submitted to CUS presented a similar profile in these parameters to that of C57Bl/6 mouse strain, we were confident to use the 129sv strain as controls of experiments performed with Alox5−/− mice.To determine the impact of 5-LO deficiency on stress-induced behavior and immune response, we began by comparing the amount of circulating corticosterone in two distinct mouse strains, C57Bl/6 and 129een them . We obseAlox5−/− exhibited significant lower amounts of circulating corticosterone and 11DHC compared to 129sv mice  = 15.70, p < 0.001; 11DHC, F = 1239.0, p < 0.001; Alox5−/− mice  = 7.98, p = 0.013; sv controls, CUS-129sv mice produced 97% more circulating corticosterone and approximately 32% less circulating ACTH  = 196.1, p < 0.001; Alox5−/− mice exhibited approximately 61% less neutrophils compared to 129sv at basal conditions  = 20.16, p < 0.01; sv mice, reaching similar levels to those of Alox5−/− mice  = 29.54, p < 0.0001; F = 20.41, p < 0.0003; sv mice. CUS exposure did not modify this profile. In contrast, CUS-129sv mice showed decreased 11DHC levels in both the frontal cortex  = 6.873, p = 0.0179; F = 45.75, p < 0.0001; sv animals.Contrasting data between blood and hippocampal corticosterone levels prompted us to perform a detailed investigation of different brain structures. Compared to 129nditions . After C−/− mice . As an aposition . Howeversv mice, 11βHSD2 activity increased in the frontal cortex of Alox5−/− and CUS-129sv mice  = 8.237, p = 0.0106; F = 12.39, p = 0.0026; sv  = 85.16, p < 0.0001; sv and in Alox5−/− mice, the reduced 11DHC levels suggest that corticosterone is produced at a higher rate than consumed, as a result of a higher 11βHSD1 activity. It is also possible that corticosterone is catabolized by a different pathway that converts it into a metabolite other than 11DHC.The 11βHSD1 and 11βHSD2 enzymes are responsible for the metabolism of corticosterone, with 11βHSD1 promoting its production, whereas 11βHSD2 converts it into the inactive metabolite 11DHC. To investigate the potential molecular mechanism regulating the balance between corticosterone and 11DHC, we assessed the enzymatic activity of 11βHSD2 in the frontal cortex and cerebellum. Compared to 129Alox5−/− and 129sv strains, submitted or not to CUS, for sucrose preference, to determine the involvement of 5-LO deficiency on that behavior. The test assesses the reduction in sugar solution intake and sucrose preference in relation to a control group  = 8.10, p = 0.007, CUS vs. strain, F = 4.68, p = 0.03; F = 5.11, p = 0.005; sv mice, which express 5-LO, develop decreased sucrose preference when exposed to CUS. However, compared to non-stressed 129sv mice, Alox5−/− animals at basal conditions already exhibited lower sucrose preference, which remained unchanged after 14 days of CUS exposure  = 8.41, p < 0.01). Supporting our previous findings, these results confirm a significant impact of 5-LO deficiency in a natural depressive-like phenotype of Alox5−/− mice that remains mostly unaltered after stressful stimuli.We evaluated exposure . These rs of CUS .CUS expo2 mediate stress responses  = 6.06, p = 0.025; F = 11.61, p = 0.002; F = 5.46, p = 0.028; sv animals, respectively. In addition, LTB4 concentration decreased by almost 25%  = 27.7, p < 0.001; Alox5−/− mice  = 18.6, p < 0.001). Compared to 129sv mice, Alox5−/− animals at basal conditions exhibited higher amounts of splenic IL-1β  = 35.94; sv mice compared to non-stressed 129sv animals but remained equally expressed in Alox5−/− mice even after CUS exposition  = 17.09, p = 0.0007) and in CUS-129sv mice  = 7.143, p = 0.0161) compared with 129sv  = 15.63 p < 0.01; sv and Alox5−/− strains expressed similar levels of 11βHSD1, which remained equally expressed in CUS- Alox5−/−, but increased significantly in CUS-129sv mice  = 27.32, p < 0.001; sv mice at basal conditions, CUS-129sv and Alox5−/− animals expressed significantly less 11βHSD2  = 22.26, p < 0.001; Alox5−/− does not seem to cause a significant impact on the expression of any of these enzymes in the adrenal gland  = 17.81, p = 0.0006; F = 10.85, p = 0.0043 respectively; The HPA axis showed increased activation in  strains . Caspaseal gland . ImportaAlox5−/− mice exhibit depressive-like phenotype at basal conditions. Excluding PGE2 production, CUS exposure does not seem to cause a significant impact on this profile, which is very similar to that of CUS-129sv mice. Considering that Alox5−/− animals lack all 5-LO-derived metabolites and LTB4 is the major product of this pathway  = 10.52, p < 0.06; F = 5.91, p = 0.016; Alox5−/− mice  which is important for the regulation of the HPA-axis and consequently corticosterone levels. This study showed that conditional Alox5−/− mice present reduced sucrose preference, suggesting reduced reward sensitivity. Furthermore, caspase-1, IL-1β, IL-17 and PGE2 increased in these animals. In frontal cortex corticosterone also increased, although its systemic levels decreased. The observed alterations were in general similar to those observed in the CUS-induced changes in control animals. In this case, however, systemic corticosterone levels increased and, notably, LTB4 levels decreased. LTB4 administration restored caspase-1 levels and systemic corticosterone levels, but not reduced sucrose preference.5-lipooxygenase (5-LO), encoded by the 4 is the main product of 5-LO activation. CUS exposure of 129sv mice, however, had a modest impact over LTB4 production in the spleen (p = 0.054). In line with these findings, reduced (CUS-129sv) or absence (Alox5−/−) LTB4 production influenced several parameters associated with stress-induced behavior, although we cannot exclude the influence of other 5-LO related metabolites. Alox5−/− animals at basal conditions already exhibited lower interest for sucrose. We also detected, using the tail suspension test (TST), that Alox5−/− mice exhibited a natural depressive-like behavior, assessed through the immobility induced by their inability or reluctance to maintain effort rather than a generalized hypoactivity. CUS exposition, however, did not change that behavior. Besides that, we observed that CUS exposure did not cause a significant impact on the immobility time of 129sv, indicating that 14 days of CUS exposure were not enough to change this behavior, contrasting with sucrose preference (data not shown). Furthermore, exposure to CUS did not impact any of the evaluated behavioral parameters in Alox5−/− mice, which were, however, similar to those observed in CUS-129sv animals. This finding shows that 5-LO-deficiency results in decreased sucrose preference that is not modified by chronic stress, possibly indicating a ceiling effect. One limitation of our study is that 129sv and Alox5−/− mice were not littermates, not sharing, therefore, the same intra-uterine and preweaning environment. This limitation is relevant because the maternal behavior could have been affected by 5-LO deletion and, consequently, affected the phenotype of the adult offspring , justifying why stress did not cause a further increase of these mediators.The basal inflammatory state of an individual is highly relevant in the context depression  in the adrenal glands. However, future studies are needed to test this hypothesis.Strikingly, we found that CUS exposition results in increased PGEh et al.  demonstrAlox5−/− mice at basal conditions, CUS-Alox5−/− show increased levels of systemic corticosterone. Treatment with LTB4 reduced circulating corticosterone but did not cause any alteration on sucrose preference. Considering that Alox5−/− mice are unable to produce other molecules beyond LTB4, we cannot discard the involvement and synergy between LTC4, D4 or E4 in sucrose preference behavior. A recent study demonstrated that the knockdown if the cysteinyl-LT receptor 1 in the hippocampus reverses the depression-like behavior of mice submitted to CMS , which is associated with the severity of depressive symptoms . The protocol was approved by the Ethics Committee on Animal Use (CEUA) of the Campus of Ribeirão Preto (Protocol number: 14.1.290.53.5).GL designed the research, analyzed the data and prepared the figures. GL and MP executed the main experimental procedures and wrote the article. KZ, PP, MB, and AP assisted with laboratory procedures. MF assisted with initial CUS protocol. LF, CS, EDB and FG analyzed the data and discussed the hypothesis. FG performed statistical analysis. LF conceived and supervised the project, designed the experiments, obtained the funding grants, helped with the data interpretation, wrote and discussed the manuscript and acted as scientific advisor.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.


Beraprost is used to treat peripheral chronic arterial occlusive disease. However, the efficacy and safety of beraprost in patients with pulmonary hypertension (PH) due to left ventricular systolic dysfunction (PH-HFrEF) remains unknown. The primary objective of this study was to determine the effects of beraprost on PH-HFrEF.We prospectively recruited patients with PH-HFrEF as determined by echocardiography and right cardiac catheterization. Beraprost sodium was given orally (1 μg/kg/d) added to the usual treatment, and patients were evaluated at 1-year follow-up.P < .05). Left ventricular ejection fraction results at 6 months (43.5 ± 7.0%), 9 months (47.0 ± 5.5%), and 12 months (48.2 ± 4.8%) were significantly higher than at baseline (34.7 ± 9.2%) (P < .05). Six-minute walking distance at 3 months (282.8 ± 80.6 m), 6 months (367.1 ± 81.2 m), 9 months (389.8 ± 87.1 m), and 12 months (395.7 ± 83.4 m) increased with time, and all were significantly higher than baseline (190.1 ± 75.5 m) (P < .05). One patient developed atrial fibrillation and recovered to sinus rhythm after intravenous administration of amiodarone. There were no instances of cardiac-related death, severe bleeding, or severe impairment of liver function.Twenty-five patients were recruited with baseline systolic pulmonary artery pressure (PAP) of 49.5 ± 10.8 mm Hg. Systolic PAP results at 3, 6, 9, and 12 months were 39.1 ± 8.1, 30.4 ± 5.2, 27.7 ± 3.0, and 27.0 ± 4.7 mm Hg, respectively, which were all significantly lower than systolic PAP at baseline (Routine oral administration of beraprost sodium added to the usual treatment may improve cardiopulmonary hemodynamics and exercise capacityin patients with PH-HFrEF. At present, treatments for pulmonary artery pressure (PAP) of PH-HFrEF patients are lacking.Pulmonary hypertension (PH) is a medically refractory disease with high cost and poor prognosis that includes 5 groups according to clinical presentation, pathological findings, hemodynamic characteristics, and treatment strategies. A small study including patients with severe primary pulmonary hypertension showed that oral administration of beraprost might result in long-lasting clinical and hemodynamic improvements. Two other studies of patients with primary pulmonary hypertension demonstrated that beraprost improved exercise capacity for up to 3 to 6 months. However, no hemodynamic improvements or long-term outcome benefits were observed.,8Beraprost is the first chemically stable and orally active prostacyclin analog, and has antiplatelet and vasodilatory effects. It is mainly used to improve intermittent claudication, pain, and cold symptoms caused by peripheral chronic arterial occlusive disease.Pathogenesis of PH-HFrEF is completely different from that of primary pulmonary hypertension, necessitating different therapeutic approaches. Few studies have evaluated use of beraprost in patients with PH-HFrEF. In this study, we administered beraprost sodium orally to patients with PH-HFrEF on a routine basis to investigate the efficacy and safety of this therapy.22.1This was a prospective pilot study conducted in an Asian country. We recruited consecutive patients hospitalized at the Second Affiliated Hospital of Soochow University China with a diagnosis of heart failure. Patients were examined by echocardiography to determine whether there was left ventricular systolic dysfunction (left ventricular ejection fraction [LVEF] <45%) and PH (systolic PAP >40 mm Hg). Patients with shock, hemorrhagic disease, severe hepatic insufficiency, acute stage myocardial infarction, pregnancy, malignant tumor, and other types of pulmonary hypertension  were excluded. Details of the study protocol were explained to the eligible patients and informed consent was obtained. The study protocol was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Soochow University.2.2Coronary angiography and right heart catheterization were performed using standard techniques for patients who were eligible for inclusion. Coronary angiography was used to determine whether patients had coronary heart disease. Right heart catheterization was used to measure mean PAP and PAWP. Inclusion criteria included mean PAP ≥25 mm Hg and PAWP >15 mm Hg.2.32) was calculated.NT-proBNP, white blood cells, hemoglobin, platelets, fasting plasma glucose, blood urea nitrogen, creatinine, and serum lipid profiles including triglyceride, total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were assessed using standard methods. Demographic and clinical characteristics of the recruited patients were collected from hospital case records and included age, sex, cigarette smoking status, hypertension, diabetes mellitus, principal diagnosis, and so on. Patient height (m) and weight (kg) in light clothing was measured, and body mass index  on a routine basis. Patients were also taking various other medications including diuretics, spironolactone, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), β-blockers, digoxin, and other drugs. If no complications, such as shock, severe bleeding, or severe liver function damage occurred, the dosage of beraprost sodium was unchanged.2.5The 6-minute walk test was given to the recruited patients at the time of selection, and at 3, 6, 9, and 12-month follow-up. Six-minute walk distance (6MWD) was recorded. Patients were accompanied by a doctor for the entirety of the test, and rescue medicine and equipment were readily available.2.6 Right ventricular (RV) systolic pressure is equal to systolic PAP in the absence of pulmonary stenosis. Systolic PAP is equal to the sum of the right atrial (RA) pressure and the RV-to-RA pressure gradient during systole. RA pressure was estimated based on the echocardiographic features of the inferior vena cava and assigned a standard value. The RV-to-RA pressure gradient was calculated as 4Vt2 using the modified Bernoulli equation, where Vt is the velocity of the tricuspid regurgitation jet in m/s.Echocardiography was performed at case admission, and at 3, 6, 9, and 12-month follow-up using a Vivid 7 ultrasound system with a standard imaging transducer. All measurements were performed following the recommendations of the American Society of Echocardiography.2.7The recruited patients were re-evaluated at least once every 3 months. Occurrences of major adverse cardiac events (MACE), such as cardiac death, heart failure readmission, nonfatal myocardial infarction, and new onset of atrial fibrillation, were recorded.Adverse drug reactions including shock, severe bleeding , and severe liver function damage were recorded during the follow-up period.2.8t tests . Data were exported to SPSS 17.0 for Windows  for analysis, and all statistical tests were conducted at the 5% level of significance.Exploratory analyses involving quantitative data were performed with independent-sample 33.1Between January, 2016 and December, 2016, 25 eligible PH-HFrEF patients, including 17 men and 8 women with an average age of 68.9 ± 7.6 years, were recruited and followed for 12 months. The hospitalization duration at recruitment was 8.6 ± 2.5 days. Baseline demographics, clinical data, and blood and echocardiographic characteristics of the study patients are summarized in Table 3.2The recruited patients were given beraprost sodium orally (1 μg/kg/d across 3 administrations) for 1 year. The dosage of beraprost sodium was unchanged since there were no occurrences of shock, severe bleeding, or severe liver dysfunction. The percentage of patients receiving β-blockers, spironolactone, or ACEIs/ARBs was greater than 60%. Nine (36%) patients underwent interventional coronary artery revascularization Table .3.3P < .05) , and showed a downward trend (3.4P < .05). LVEF at 3 months was 37.6 ± 8.2%, which was not significantly different from baseline. After a year of treatment, the LVEF was generally increasing , 9 months (47.0 ± 5.5%), and 12 months (48.2 ± 4.8%) was significantly higher at each point than at baseline (34.7 ± 9.2%) (3.5P < .05) , 6 months (367.1 ± 81.2 m), 9 months (389.8 ± 87.1 m), and 12 months (395.7 ± 83.4 m) months increased gradually, and all distances were significantly higher than the baseline value (190.1 ± 75.5 m)  or severe impairment of liver function.4This pilot study prospectively evaluated the efficacy and safety of beraprost in patients with PH-HFrEF. The results showed that routine oral administration of beraprost sodium added to the usual treatment reduced systolic PAP, and improved LVEF and 6MWD. There were no instances of cardiac death, myocardial infarction, shock, severe bleeding, or severe impairment of liver function, which indicated good efficacy and safety of beraprost in these patients.,11 PH is prevalent in patients with chronic heart failure. Up to 60% of patients with severe left ventricular systolic dysfunction may also have PH, and almost all patients with severe symptomatic mitral valve disease have PH with or without heart failure.,13 Despite appropriate treatment, patients with PH-HFrEF still have a high 5-year readmission rate and all-cause mortality rate.Pulmonary hypertension due to left ventricular systolic dysfunction is different from other types of PH with regard to pathogenesis and management. Backwards transmission of filling pressures due to impaired left ventricular systolic function is the main cause of PH-HFrEF, along with high PAWP. Unlike heterozygous BMPR2 mutations, which account for approximately 75% of familial pulmonary arterial hypertension cases, no specific genetic linkage has been identified for PH-HFrEF. Most clinical findings showed that PH-specific therapies  had no effect on PH-HFrEF, and some may even be harmful.–17 General therapies for heart failure including diuretics, digoxin, ACEIs, and β-blockers are recommended for treatment of PH-HFrEF. Diuretics are the main medical treatment for relief of fluid load and congestion, and ACEIs and β-blockers improve prognosis. Furthermore, identifying and treating possible causes of heart failure, such as coronary artery disease, cardiomyopathy, and heart valve disease, is important.Management of PH-HFrEF is a systematic process. Treatment of group 1 PH  in the ESC/ERS guidelines does not apply to patients with PH-HFrEF.,21 In animal models and human trials, prostacyclin decreased PAP and improved function of the right heart in right ventricular failure because of its vasodilatory effects.,23 However, a randomized controlled trial of epoprostenol infusion for treatment of severe congestive heart failure yielded different results. This trial was terminated early because of a strong trend of decreased survival in patients treated with epoprostenol. Intravenous epoprostenol was not associated with improvement in 6MWD or quality of life, but was associated with increased risk of death.Whether prostacyclin and its analogs can be used in patients with heart failure is controversial. Several studies of intravenous prostacyclin in patients with left ventricular systolic dysfunction after cardiac surgery showed that prostacyclin could decrease mean PAP, PAWP, and systemic and pulmonary vascular resistances, and increased cardiac output was comparable to that resulting from sodium nitroprusside or inhaled nitric oxide treatment. Like prostacyclin, beraprost acts on the prostacyclin receptor in platelets and vascular smooth muscle, activates adenylate cyclase, increases intracellular cyclic adenosine monophosphate concentration, inhibits Ca2+ influx and thromboxane A2 production, and thus induces antiplatelet and vasodilatory effects. In addition, beraprost can increase endothelial nitric oxide synthase expression and nitric oxide production in murine and bovine aortic endothelial cells, and suppress TNF-α expression in human monocytes via mitogen-activated protein kinase pathways, all of which improve vascular diseases. Previous studies of beraprost for treatment of pulmonary hypertension evaluated group 1 PH in the ESC/ERS guidelines.–8 Although a small study with 13 patients with severe pulmonary arterial hypertension demonstrated that beraprost may result in long-lasting clinical and hemodynamic improvements, 2 randomized controlled trials with more than 100 patients with pulmonary arterial hypertension confirmed that beraprost only improved exercise capacity, which persisted for up to 3 to 6 months, but did not improve long-term prognosis.,8 No obvious side effects were observed.Beraprost is a stable, orally administered prostacyclin analog recommended for treatment of chronic peripheral arterial disease.,28Pulmonary hypertension due to left ventricular systolic dysfunction is completely different from group 1 and other types of PH in both pathogenesis and treatment. This study was the first to use beraprost in patients with PH-HFrEF, regardless of dosage and duration. Based on beneficial effects of beraprost on vessel dilatation and antiplatelet activity, routine oral beraprost treatment was hypothesized to provide therapeutic benefit to patients with PH-HFrEF, which was confirmed in this pilot study. Several studies have shown that beraprost can inhibit myocardial apoptosis and myocardial fibrosis, which is 1 of the possible intrinsic mechanisms for beraprost improving the prognosis of PH-HFrEF patients.Major side effects of beraprost include severe bleeding, hypotension, and shock. In patients with PH-HFrEF, therapeutic strategies typically include combinations of diuretics, ACEIs, and antiplatelet drugs, which may increase the incidence of side effects. Therefore, when side effects occur, it is important to evaluate which drugs are responsible. Moreover, follow-up frequency should be increased. Once the cause of side effects is discovered, the type and dosage of the drugs should be adjusted accordingly. In this study, beraprost sodium was administered at 1 μg/kg/d and was not titrated to a larger dose. If bleeding or shock had occurred, the cause would have been investigated, and the dosage of beraprost sodium or other drugs would have been adjusted. In this study, a patient experienced hematochezia, which was caused by hemorrhoids. After adjusting the aspirin dosing interval, hematochezia stopped.4.1Our study had the following limitations. The small sample size precluded making conclusive statements regarding our results. In addition, follow-up PAP results obtained by echocardiography were not as accurate as right cardiac catheterization. Furthermore, this study was an observational study, and no control group was evaluated. As such, more randomized controlled studies are needed to investigate the efficacy and safety of beraprost in patients with PH-HFrEF.4.2A multicenter randomized controlled clinical trial (ChiCTR-IPR-17012961) with larger samples designed based on the results of this pilot study is in progress and we will detect the dynamic changes of NT-proBNP in this study. In the near future, we will have more evidence for clinicians.5Routine oral administration of beraprost sodium added to the usual treatment may further decrease PAP, and improve LVEF and 6MWD in patients with PH-HFrEF, indicating cardiopulmonary hemodynamic and exercise capacity benefits. Meanwhile, treatment with beraprost exhibited good safety.The authors gratefully acknowledge the assistance of Miss Yan-Ni Wu and Jing Zhu with patient recruitment.Conceptualization: Yuqi Chen.Data curation: Xinyi Zhu, Yuqi Chen.Formal analysis: Xinyi Zhu.Funding acquisition: Liang-Ping Zhao.Investigation: Maosong Wang, Xiang Liu.Methodology: Xinyi Zhu, Maosong Wang, Xiang Liu.Project administration: WeiTing Xu.Supervision: JiangChang Chen, WeiTing Xu.Validation: Maosong Wang, Yuqi Chen.Writing – original draft: Li Wang.Writing – review & editing: Liang-Ping Zhao.


Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ Binge alcohol drinking  is increasingly popular among young people, and is especially damaging to the embryo during the third to fourth weeks of pregnancy, when most pregnancies are unrecognized3. Alcohol exposure during this period causes characteristic craniofacial malformations of Fetal Alcohol Syndrome (FAS) including small palpebral fissures , a smooth philtrum, and brain malformations in the holoprosencephaly spectrum3.Fetal Alcohol Spectrum Disorder (FASD) is common; recent conservative prevalence rates range from 1–5%4, and about 4% of pregnancies are marijuana-exposed5, through either recreational use or as an anti-nausea self-medication. However, some subgroups of pregnancies have considerably more exposure7 and nearly 20% of a cohort of pregnant women in California, aged 18–24, report marijuana use8. As marijuana and other cannabinoids (CBs), such as cannabidiol (CBD), become increasingly legalized for medical or recreational purposes, and they remain perceived as low-risk substances9 safe to use during pregnancy, the incidence of CB-exposed pregnancies will rise even further. A recent study of births in Colorado found that the incidence of several birth defects has risen in the state during the period of marijuana legalization10.Marijuana use is also risingCannabis derived CBs, include the synthetic CBs, which can be 100-fold more potent for CB receptors than is THC11, and the endocannabinoids (ECBs) which have several physiological functions, especially during development12. Perturbation of the ECB system by embryonic and/or fetal CB exposure has several developmental consequences. Before implantation, CBs can delay early embryo development and implantation13 and marijuana use has been associated with spontaneous abortion and pre-term birth, particularly when used with other drugs14. Limited studies in the 1970s and 1980s revealed that some prenatally-exposed individuals have physical and psychological differences resembling FASD16. Since today’s marijuana is at least 4-fold more potent17, it is likely that the consequences of prenatal CB exposure are more severe than first thought. Synthetic CBs, in particular, may pose greater harms to the embryo.CBs are a diverse family of compounds and in addition to the 23. Previously, we found that a single, neurulation-stage exposure to the synthetic cannabinoid CP 55,940 dose-dependently induced craniofacial and brain abnormalities, mostly in the holoprosencephaly (HPE) spectrum24, and similar to a high-dose alcohol exposure3. During neurulation , the neural tube closes, and the eyes and brain form out of the neuroepithelium. As predicted by these developmental events, CP 55,940 caused significant brain and eye defects, ranging from mild microphthalmia to severe anophthalmia, which are also common in FASD25. The sensitivity of the neurulation-stage embryo to many CBs supports clinical studies demonstrating that first trimester marijuana exposure, the most common exposure26, has developmental effects29.Animal studies, precisely controlling drug exposures, corroborate the morphological and life-long behavioral effects of both phyto- and synthetic-cannabinoids31. Combined alcohol and CBs is significantly more impairing than is either substance alone and simultaneous use is associated with a greater risk for negative health outcomes32. Unfortunately, the combined effects of alcohol and CBs during pregnancy have not been systematically studied33. Boa-Amponsem et al.34 have recently demonstrated that combined exposure to a CB1 receptor agonist, ACEA, and ethanol worsen embryonic dysmorphology and juvenile behavioral phenotypes in zebrafish, in a manner reversible by either CB1 receptor antagonism or the addition of Sonic hedgehog (Shh) mRNA. Here, using mouse and zebrafish models of drug exposure during neurulation, we evaluated how simultaneous exposure to alcohol and a variety of CBs affects embryonic development and established a pathogenic mechanism involving a novel interaction between the CB1 receptor and the Shh signaling pathway. While prior studies implicate Shh signaling in alcohol teratogenesis39, less is known about how CBs affect Shh signaling. Recently, ECBs and phytocannabinoids (i.e. THC and CBD) were shown to inhibit Shh signaling in vitro by direct interactions with Smoothened (Smo)40. Smo, a G-protein-coupled, seven-pass transmembrane protein, regulates PKA levels and the activity of GPR161 within the primary cilium42 which affects the processing of downstream glioma associated oncogene (Gli) transcriptional activators. We now report that in the embryo, Smo and CB1 form heteromers that are likely targets of the teratogenic effects of simultaneous alcohol and CB exposure.CBs are frequently used with alcohol, partly to intensify their individual psychoactive effects, but also because one substance can disinhibit the use of the other24 to other CBs. Pregnant C57 mice received a single intraperitoneal injection of the synthetic cannabinoid HU-210, the phytocannabinoids CBD, or Δ9-THC, or the CB vehicle on their 8th day of pregnancy (GD 8 – the beginning of neurulation). While THC concentrations vary between cannabis strains and preparation, the mouse THC doses (0.56–17.0 mg/kg) replicate peak blood levels in the range that frequent cannabis users can achieve in a controlled laboratory setting49. The CBD doses administered in this study (1.7–17 mg/kg) are within the therapeutic range (<1–50 mg/kg/day) for several medical conditions50. Eye defects were evaluated using a dysmorphology scale modified from51 , the CB1 agonist examined in our previous teratology study24, because its effects are highly dose-dependent without being embryonically lethal. CP 55,940 did not increase embryonic cell death, ruling this out as a primary pathogenic mechanism  exposure, we collected embryos with 15–16 somites, dissected out the rostral neural tube (which will form the brain and the face), and processed them individually with real-time quantitative PCR for the expression of Shh, Gli1, and Gli2 mRNA, relative to the housekeeping gene 18s. CP 55,940 significantly reduced Shh and Gli1 gene expression, relative to stage-matched vehicle-treated embryos . This amount of N183 mRNA had no detectable effects on eye or brain morphology when given alone . In zebrafish, we also administered SR 141716A before the combination of a low CP 55,940 dose (2.5 mg/L) and the sub-teratogenic alcohol dose (0.5%) previously found to potentiate each other’s effects  attenuates CP 55,940 teratogenesis. We focused on the CB1 receptor because CB1 is expressed during early neurulation, but CB2 is not detectablesee Fig. . Alone, see Fig.  and the see Fig. . Thus, i57. CB1 and Smo, at least in drosophila, each can form homodimers to maximize their effectiveness at altering cell signaling60, and CB1 can heterodimerize with many other GPCRs including: CB261; mu62 and delta63 opioid receptors; 5-HT2A64; angiotensin II65; orexin66; D267; adenosine 2A68; and GPR5569. In the case of CB1-D2L heteromers, agonist binding can shift G-protein signaling from Gαi to Gαs70, and cause an unexpected increase in PKA levels. In the primary cilium, where canonical Shh signaling takes place, PKA activity is reciprocally regulated by Smo and GPR 16171. To ascertain whether CB1 receptors directly associate with Smo, we performed a proximity ligation assay (PLA) in GD 9.5 embryonic neural tube tissue to visualize CB1 and Smo antibodies bound within 40 nm of each other  weighing ca. 20 g were mated to males of the same strain for one to two hrs beginning at least two hrs into the light phase of a 12:12 light:dark schedule. When a copulatory plug was found, gestational day (GD) 0 was designated as the start of the mating session. Mated females were housed in clear, ventilated, polycarbonate cages with no more than three mice per cage, depending on the number of plugs detected in a mating session. Food  and water were freely available, housing and nesting material were provided, and the cob bedding was changed every other week. The vivarium was maintained at 21 ± 1 °C, 30–40% humidity. All mouse experiments followed NIH guidelines using methods approved by the IACUC of UNC at Chapel Hill. Zebrafish were obtained from Zebrafish International Resource Center. The AB strain was used in this study and fish were housed in automatic fish housing systems  at 28.5 °C. All procedures using zebrafish were approved by the North Carolina Central University IACUC and conducted according to NIH and U.S. regulations.9-THC  and dissolved in deoxygenated 100% ethanol . CB aliquots were prepared immediately before injection by suspension in alkamuls EL 620  and dilution to 5% ethanol, 5% alkamuls in lactated Ringer’s solution (i.e. 1:1:18). Control mice were injected with an equivalent volume of the CB vehicle solution, which provides a 0.59 g/kg dose of alcohol and causes peak maternal blood alcohol levels of 43.8 mg/dl24.On GD 8, dams were weighed and injected intraperitoneally with freshly prepared drug or vehicle solutions through a 27-gauge needle at a volume of 1.5 ml/100 g body weight. For CB alone studies, the mice received a single injection of either 0.03 or 0.1 mg/kg of HU-210 -6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzopyran-9-methanol), 1.7, 5.6, or 17 mg/kg cannabidiol (2-[1R-3-methyl-6R-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol), or 0.56, 1.7, 5.6, or 17 mg/kg of Δcis-3-[2-hydroxy-4-phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol, Tocris Bioscience, Minneapolis, MN) suspensions were diluted to 23.7%, 11.85%, or 5% ethanol and 5% alkamuls to administer the 2.8 g/kg, 1.4 g/kg alcohol doses, or the CB vehicle doses, respectively. The mice received a second injection of either the CB vehicle or the appropriate alcohol dose in the CB vehicle, four hrs after the first injection. No additional CB dose was given in the second injection. The purpose of the second injection was to follow established procedures for inducing birth defects following early gestational acute alcohol exposure3. Peak blood alcohol levels following the second 2.8 g/kg dose are 381 mg/dl80 and 132.2 mg/dl following the 1.4 g/kg dose , THC (0.56 mg/kg) or CP 55,940 - mg/kg, T2 and cardiac blood was collected. Alcohol levels were measured enzymatically by an Analox alcohol analyzer .Blood alcohol levels were measured in GD 8 dams (n = 5/treatment) injected with alcohol (1.4 g/kg) alone, or alcohol combined with CP 55,940 (0.25 mg/kg), and all mice received a second alcohol injection, 4 hrs later. 30μl of tail blood was collected 30 min after the first alcohol injection, and 30, and 60 min after the second alcohol injection. 2 hrs after the second alcohol injection, the mice were euthanized with COFor the CB1 receptor antagonist study, SR 141716A (5-(4-chlorophenyl)-1--4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide) was obtained from Tocris Biochemicals , prepared as above, but diluted to a 5% ethanol, 5% alkamuls suspension in lactated Ringer’s (i.e. 1:1:18). SR 141716A (2.0 or 5.0 mg/kg) or the CB vehicle was administered 10 min before CP 55,940 (0.5 mg/kg) or the CB vehicle. In this experiment, the volume of each injection was 0.75 ml/100 g to ensure that the dams received comparable amounts of alcohol from the CB vehicle (i.e. 0.59 g/kg) as in our other dose-response studies.2 inhalation and euthanized by cervical dislocation. The uteri were removed, and the fetuses were dissected free under ice-cold phosphate buffered saline (PBS). Fetuses were individually examined for body weight, crown-rump length, gross dysmorphology, and the presence of eye defects, evaluated on a seven-point dysmorphology scale  with a Micropublisher 5.0 digital camera and QCapture Suite software . After being photographed, the fetuses were immersion fixed in Bouin’s solution or 10% formalin. Select fetuses with eye and/or facial malformations were processed for routine histological analyses. Bouin’s-fixed heads were cleared in 70% ethanol, embedded in paraffin, and sliced coronally at 10 μm using a rotary microtome. Serial sections were mounted on glass slides and stained with hematoxylin and eosin (H&E).Fetal dysmorphology measurements were taken on GD 17. Dams were deeply anesthetized with CO81, was performed in embryos treated with the CB vehicle (n = 9 embryos from 3 litters) or 0.5 mg/kg of CP 55,940 (n = 8 embryos from 3 litters) at GD 8. Twelve hrs after the injection (GD 8.5), the embryos were dissected in lactated Ringer’s solution at 37 °C, freed from all extra-embryonic tissues, transferred to a 37 °C Nile Blue solution , and occasionally swirled for 30 min. Following a brief rinse in lactated Ringer’s, the embryos were immediately photographed, as above.Nile blue sulfate staining, modified from82 and involved measuring the longest axis along the eye, calculated against a standard 50-µm ruler under the same magnification. For the 48 hpf eye, we designated a diameter less than 240 µm as having a small eye phenotype, because untreated eyes were typically at least 250 µm in diameter. All the surviving embryos from different treatments were collected at 24 hpf and/or 48 hpf, and fixed in 4% paraformaldehyde in PBS.Zebrafish embryos were incubated in 100 mm plates containing 10 to 20 embryos in fish water  containing a 1:500 dilution of 0.1% methylene blue . Embryos were exposed from 5.25 to 48 hrs post fertilization (hpf) to CP 55,940 dissolved in DMSO and diluted in fish water to 1 mg/L, 2.5 mg/L, 3.75, or 5 mg/L. DMSO was added to the fish water as control for CP 55,940. Ethanol was diluted in fish water to either 0.5% or 2%. At the end of treatments, fish water containing CP 55,940 and/or ethanol was removed, and embryos were washed once with fresh fish water. Malformation of the MHB was assessed visually, based on the absence of the defined border between the midbrain and hindbrain at 24 hpf stage see Fig. . Eye siz82. For the CB1 receptor antagonist study, SR 141716A was dissolved in DMSO and added to fish water at a concentration of 3 mg/L 30 min before CP 55,940 exposure.For the ShhN183 mRNA injection study, one to two cell stage embryos were injected with 25 pg of capped ShhN183 mRNA. N-terminal 183 amino acid zebrafish Shh (ShhN183) was synthesized by using 22 hpf zebrafish embryo mRNA as template and oligo dT as primer, using Superscript reverse transcriptase. PCR primers were CGGAATTCATGCGGCTTTTGACGAGAGTG and GCTCTAGATCAGCAATGAATGTGGGCTTTGG. The PCR product was subcloned into pCS2 vector and capped mRNA was synthesized with Ambion cap mRNA kitCannabinoid compounds including CP 55,940 , cannabidiol, and HU-210  were prepared as 25 mM stocks in deoxygenated DMSO supplemented with 0.1 mg/mL fatty acid-free BSA . The Smoothened agonist, SAG (N-methyl-n′-(3-pyridinlybenzyl)-n′-(3-chlorobenzo[b]thiophen-2-caronyl)-1,4-diaminocyclohexane, Santa Cruz Biotechnology, Dallas, TX) was prepared in DMSO.83. First, the firefly luciferase assay buffer was added using the 384-dispense head fluidics  and the firefly signal was read using a luminescence plate reader . Next, the renilla assay buffer was added to quench the firefly signal and read the renilla signal. Data were expressed as % change in the firefly:renilla ratio from the maximal SAG alone effect, and plotted using a sigmoidal curve fit.Shh-L2 cells  were plated using the Mutidrop-384 (ThermoFisher) at 3000 cells per well in 384-well plates in complete growth media . After the cells reached 100% confluency (48 hrs later), the media was changed to induction media . The Biomek NX workstation  was used to remove and add media. CB treatments were performed at the following concentrations  in the presence of randomized triplicate SAG concentration responses (up to 0.5 µM). For the CP 55,940 concentration response, a maximally effective 300 nM SAG concentration was added to the induction media. Randomized triplicate CB dose responses were then conducted using the D300 digital dispenser . For controls, stimulated compound vehicle wells were included to determine baseline stimulation and ‘no SAG’ wells to calculate fold induction. After 48-hrs incubation with the compounds, 30 µl of the media was removed (20 µl left) and the dual luciferase assay was conducted following a published protocolTo determine the effects of CBD, CP 55,940, and HU-210 on the viability of the Shh-L2 cells, 384-well plates were seeded with 700 cells per well to a total volume of 45 uL using the Multidrop 384 microplate dispenser and cells were allowed to attach overnight. Cells were treated with CBs or DMSO vehicle control and cultured for a further 72 h. Media was removed and replaced by pre-warmed dye cocktail in PBS . Plates were then returned to the incubator for 45 min, washed and then fixed by adding 20 uL 10% formalin for 15 min. Plates were washed with PBS and then sealed for imaging. Fluorescence quantification and localization was determined using a ThermoFisher CellInsight NXT and 3-channel Cell Health Profiling protocol in HCS Suite software (ThermoFisher). Excitation wavelengths were 386 nm, 485 nm, and 549 nm for Hoechst 33342, YOYO-1, and MitoTracker Red FM, respectively. Fixed exposure times were optimized in each channel for each experiment and set so that camera pixel intensity saturation was not reached. Images were acquired using an Olympus UPlanFLN 10 × /0.30 objective and 2 × 2 camera binning. A nuclear mask was established using channel 1 signaling (Hoechst 33342) and used to determine nuclear characteristics  as well as establishing the regions of interest for YOYO-1  and MitoTracker Red FM . Data analysis was performed retrieving data sets from HCS View software (ThermoFisher) and normalizing to mean DMSO values.84. The cells attached overnight resulting in 50% confluency for treatment the day after plating. For CB dose-response studies, cells were stimulated with the previously determined EC90 dose of SAG (100 nM). CBs were then added to treatment wells using a D300 digital dispenser  at 1:2 or 1:3 serial dilutions in triplicate. Intra-assay controls included DMSO-treated and SAG-stimulated wells alone, and “no cell” wells for background subtraction. Plates were then sealed with gas permeable, liquid impermeable membranes  and incubated under humidified conditions at 37 °C and 5% CO2. After 5 days of treatment exposure, the medium was removed from the wells and replaced with a brief 37 °C PBS wash. The PBS was removed and 1X pNPP alkaline phosphatase substrate  in diethanolamine  was added in a volume of 100 µL per well as per the manufacturer’s instructions. Plates were incubated at room temperature in the dark for one hr and colorimetric development was evaluated at 405 nm by SPECTRAmax PLUS 384 . Data analysis was performed by subtracting “no cell” averages within the same plate from all treatment wells and presenting as percent inhibition vs. DMSO control for plots generated in GraphPad Prism . Plotted data were fit to a sigmoidal curve.C3H10T1/2 cells  between passages 5 and 15 were plated in 96-well plates at 5000 cells per well from sub-confluent propagation flasks in Dulbecco’s Eagle’s high glucose medium , supplemented with 10% bovine calf serum , 2 mM L-glutamine , and 100 units/ml penicillin/100 units/ml streptomycin  following previously published methodsShh (Mm00436528), Gli1 (Mm00494654), and Gli2 (Mm01293117). 18 s (Mm03928990) was used as a reference gene. All reactions for each gene target and reference were run in triplicate. Product specificity of the amplicon was assessed using ethidium bromide-agarose gel electrophoresis. The comparative Ct method was used to obtain the relative fold change in gene expression of experimental (CP) vs. the average of vehicles per plate85. Data were transformed and expressed as the log2 fold change from the vehicle group.Dams were euthanized as above, 24 hrs after injection with either the CB vehicle or CP 55,940 (0.25 mg/kg). The embryos were quickly dissected from the placenta in chilled RNAse-free Dulbecco’s solution. After counting the number of somites for developmental staging, the headfolds were removed and rapidly frozen on dry ice and stored at −80 °C. The tissue was homogenized via vortexing in buffer RLT plus . RNA was extracted using the RNeasy plus micro kit  and stored at −80 °C. Nucleic acid concentrations and quality were determined using fluorimetry  and spectrophotometry . cDNA was synthesized from the RNA using the Quantitect reverse transcription kit . Gene expression was assessed using real-time quantitative PCR  with Taqman probes  for the following target genes: in situ probes. Two control antibody combinations were also used to test the specificity of the probes: (1) CB1 and Sox-2 , to test whether CB1 antibody labeling, which should in the membrane or cytosol, would proximity label with a nuclear bound transcription factor; and (2) Smo and Arl13b , to test whether Smo would proximity label with a primary cilia bound protein. Otherwise, processing was identical to the parameters described above. Embryos were imaged on a Zeiss 880 confocal microscope with a 40x or 63x oil lens.On GD 9.5, pregnant C57BL/6 J dams  were euthanized as above and embryos were quickly dissected from the placenta and decidua, placed into 4% paraformaldehyde in 1X phosphate-buffered saline (PBS), and were stored for one week at 4 °C. Somites were counted to stage-match embryos (24–25 somites) for further processing beginning with paraffinization on a Leica TP1020 tissue processor ; samples were dehydrated with a graded ethanol series, cleared with toluene, and infiltrated with paraffin (Paraplast Plus). Once infiltrated, the embryos were embedded in paraffin and serially sectioned at 7 μm on a microtome. Three slides (4–5 sections/slide) including tissue from the neural tube were chosen and processed using DuoLink PLA Technology  according to the manufacturer’s instructions. Briefly, slides were deparaffinized, rehydrated with a graded ethanol series, placed in a heated citric acid buffer for 30 min, blocked with blocking buffer supplied in the kit for 1 hr, then incubated for 36 hr with either an antibody cocktail  in antibody diluent or antibody diluent alone as a no-primary control. Sections were then processed according to the kit’s instructions using the appropriate 2, 20% glycerol) with protease inhibitor cocktail  in a ratio of 1:30 (v:v) following methods described previously87. An affinity resin for the anti-CB1R or anti-Smo was prepared by coupling affinity-purified anti-CB1R or anti-Smo antibodies to Affi-Prep-Hz matrix  according to the manufacturer’s instructions. This method binds periodate-oxidized carbohydrate moieties on the antibody heavy chain to hydrazide-activated methacrylate matrix88. The CHAPS solubilized proteins (~10 µg/embryo sample) were precleared by mixing with protein A/G coated sepharose bead without any antibody (1:1 volume ratio) for 30 min at 0–4 °C and centrifuged at 1000 × g for 5 min. Supernatant was collected as a precleared sample and then mixed with anti-Smo  or anti-CB1R  affinity resin in a total volume of 100 µl (volume adjusted using solubilization buffer) and incubated for 3 hrs at 0–4 °C with constant rotation mixing. Affinity matrix bound immunoprecipitated proteins were spun down (1000 × g for 5 min), washed twice with NaCl/Tris . Immunoprecipitated proteins were eluted from the matrix with 200 µl glycine buffer  and eluate was immediately neutralized with Tris/HCl . The proteins from the neutralized eluate were then precipitated by adding 8 volumes of CHCl3/CH3OH/H2O (1:4:3), dissolved in Laemmli’s sample buffer containing 5 mM EDTA, and heated at 65 °C for 5 min. Proteins were resolved in 10% SDS-PAGE, followed by Western blot and ECL detection, and probed with Gαs antibody , or anti-CB1R polyclonal . Immunoblots were visualized using a chemiluminescent reagent . Bio-Rad GelDoc XR+ along with Image Lab Software 5.2.1  was used to capture the images. All images were captured using consistent, standard settings and multiple images were captured at 5 sec intervals for 30–40 sec to track the saturation of the bands. Optimum images (without any oversaturation) were analyzed using Image J software89.GD 8 dams were injected with either the CB vehicle (n = 2), 2.8 g/kg alcohol (n = 2), 0.25 mg/kg CP 55,940 alone (n = 2), or with 2.8 g/kg alcohol (n = 2). Four hrs later, they received a second injection of the CB vehicle, or 2.8 g/kg alcohol. 30 min after the second injection, dams were euthanized as described above and whole embryos were collected, pooled as a litter (to achieve adequate protein concentrations for immunoprecipitation), rapidly frozen on dry ice, and stored at −80 °C. The samples were lysed and solubilized in CHAPS buffer  as the primary antibody and did not exhibit bands appearing in the range of CB1R receptors or G proteins. To control for the immunoprecipitation procedure, the washings following immunoprecipitation were run on the gel; no leakage of either CB1R receptor or G-proteins was observed. We performed a mock immunoprecipitation of the extract over agarose that was lacking the attached antibody. No CB1 receptor or G-protein were eluted from the mock matrix material.The following control experiments optimized the immunoprecipitation conditionsShh pathway gene expression was analyzed with unpaired t-tests vs. the CB vehicle group. Incidence data are expressed as the proportion of mice or zebrafish with a specific defect . All other data are expressed as mean ± SEM except where otherwise stated. Statistical significance was set as an alpha level of 0.05.The incidence of eye defects in mice and fish was analyzed with Chi-square tests comparing two specified treatments, either to the CB vehicle control or the cannabinoid alone. Litter size, body weights, crown-rump lengths, and blood alcohol levels were analyzed with one-way analysis of variance (ANOVA) tests and post-hoc Bonferroni comparisons vs. the CB vehicle group as appropriate. Supplemental Figures and Tables


According to the experiment results, both copolymers possessed outstanding film-forming ability, which cannot be realized by small PDI molecules and oligomers. And they can serve as a superior candidate as for n-type materials, especially for their relatively wide range of light absorption (λ = 200~800 nm), and lower LUMO level (−4.3 and −4.0 eV).In order to improve the poor film-forming ability of polymeric ladderphane, di-block copolymers containing perylene diimide (PDI)-linked double-stranded poly ladderphane and branched alkyl side chains modified single-stranded poly were synthesized by metathesis cyclopolymerization (MCP) using Grubbs third-generation catalyst (Ru–III) in tetrahydrofuran solvent. The first block containing the ladderphane structure leads to higher thermal-stability, wider UV–vis absorption, lower LUMO level and ladderphane-induced rigidity and poor film-forming ability. The second block containing long alkyl chains is crucial for the guarantee of excellent film-forming ability. By comparing the effect of ladderphane structure on the resulted copolymers, single-stranded poly derivatives with PDI pedant were also processed. The structures of copolymers were proved by  It is well-known that perylene diimide (PDI) chromophores are widely used for constructing n-type organic semiconductors, as PDI derivatives generally show high thermal and chemical stabilities, good electron-accepting abilities, and excellent electron mobilities ,2,3,4,5.Great progress has been made on a new class of polymers, which was defined as polymeric ladderphane comprising of double stranded polymeric skeleton with multilayer planar oligoaryl linkers. More precisely, a linear array of co-facially oriented arenes with polymeric backbones as the tethers ,8,9,10. DDDPM with two long alkyl tails was synthesized, and then was copolymerized with PDI bridged bifunctionalized 1,6–heptadiyne monomer 1 , copolymerization of 1 and comonomer DDDPM bearing longer alkyl chain was supposed to be an attractive method to prepare soluble diblock copolymer, and therefore the copolymerization of 1 and didodecydipropargylmalonate (DDDPM) –b–Poly(DDDPM) with a narrow molecular weight distribution (PDI = 1.3) in 98% yield (Run 1). At a higher monomer loading, the higher [1]/[DDDPM]/[I] ratios of 20:80:1 and 40:80:1 (Runs 2~3) were attempted and expected to obtain higher molecular weight polymers, whereas the isolated polymer yields reduced to 76%, but there were two molecular weight values showing that some propagating carbenes discomposed when the polymer backbone increased to a certain size /[DDDPM]/[I] ratio of 40:80:1, produced the polymer with a little wider PDI values (1.5) in 70% yield.For improving the solubility of poly( (DDDPM)  was carrain size ,15. Simi3, CH2Cl2, THF, chlorobenzene, and xylene), and had good film-forming properties. For example, when solutions of the copolymers in CH2Cl2 were added dropwise onto a slice of glass, and then after solvent evaporation, a black-green shiny film (DDDPM) block to poly(1), which indicating its great potential for solution processed organic devices and circuits. Similar results were also found for Poly(2)–b–Poly(DDDPM).Both the resultant copolymers were easily soluble in common organic solvents  and terminal alkyl group on DDDPM (DDDPM]/[Ru–III] (20/80/1). Thus, this result confirmed the successful attachment of poly(DDDPM) to the poly (1) block, while, as can be seen from 2)-b-Poly(DDDPM) were derived from perylene core and methylene protons adjacent to nitrogen. There is no unique peak attributed to Poly(DDDPM). However, the integral ratio of methylene group and methyl groups on Poly(2)–b–Poly(DDDPM) changed from 4:3 to 8:3. This obvious increase was caused by the introduction of the second block Poly(DDDPM). Because there are two dodecyl groups on the DDDPM molecule, which contain many methylene group, thus, the increase of methylene group content in the copolymer is due to the introduction of DDDPM segments. The two blocks contain multiple methylene and methyl groups simultaneously, the content of the two blocks cannot be calculated by 1H NMR data. Meanwhile, it was found that GPC curves showed only one peak, and the molecular weight distributions of the diblock polymers were narrow, hence there were no homopolymer mixtures in the block polymer by GPC characterization (occurred . Moreoveon DDDPM . Coincidrization .ELUMO = −(4.65 + 1)–b–Poly(DDDPM), −4.0 eV for Poly(2)–b–Poly(DDDPM), lower than the well-studied PDI derivative (−3.8 eV) dichloro(benzylidene)bis(3–bromopyridine)ruthenium(ii)  and 1–dodecanol were obtained from Aldrich. Ethyl vinyl ether , EDCI·HCl  and DMAP  were added under nitrogen atmosphere in ice-water bath and stirred for 2 h, then the reaction progress proceeded at room temperature and was monitored by TLC. After 6 days, the mixture was washed by dilute hydrochloric acid (5 × 30 mL), followed by water, and dried with anhydrous MgSO4. After filtration and removing the solvent, the crude product was purified by column chromatography on silica gel using 1:20 CH2Cl2/petroleum ether as eluent. DDDPM was obtained as a white waxy solid . 1H NMR : δ 4.20–4.12 , 2.65 , 2.02 , 1.45–1.7 , 0.94–0.80 ; 13C NMR : δ 178.28, 80.2, 70.5, 64.58, 50.6, 31.7, 29.6, 29.3,29.1, 25.8, 22.9, 22.4, 14.3; ESI–MS: Calcd. For C33H56O4Na [M + Na]+: 539.4176, Found: 539.4169.2,2–bis(prop–2–yn–1–yl)malonic acid  was firstly dissolved in 50 mL of anhydrous CH1  and Ru–III  were stirred in 80 mL of THF at 30 °C. After it had been confirmed that 1 had disappeared by TLC (nearly 20 min), the 1 mL solution of the second block  was added to the Schlenk tube, the reaction mixture was stirred at 30 °C for another 3 h. After being quenched by adding excess ethyl vinyl ether, the concentrated reaction mixture of Poly(1)–b–Poly(DDDPM) was precipitated to acetone, and the purple–black solid was washed with acetone till the filtrate was colorless, and then was dried in a vacuum oven at 40 °C to a constant weight.Typically, polymerization was carried out in a Schlenk tube under dry nitrogen atmosphere at 30 °C in THF for a preset time. Monomer 2)–b–Poly(DDDPM) was similar to that of Poly(1)–b–Poly(DDDPM), except for the initial concentration of monomer 2. Monomer 2  and Ru–III  were stirred in 1 mL of THF at 30 °C. After it had been confirmed that 2 had disappeared by TLC (nearly 2 h), the 1 mL solution of the second block  was added to the Schlenk tube, and the reaction mixture was stirred at 30 °C for another 3 h.The preparation of Poly(Eg of Poly(1)–b–Poly(DDDPM) and Poly(2)–b–Poly(DDDPM) can even narrowed to 1.55 eV, and the LUMO energy lowered to −4.3 and −4.0 eV, respectively, which shows prospective application in solar cell and other devices. Based upon this study, further investigation of the application in photoelectric devices is still under way.In this study, diblock copolymer ladderphane containing PDI group was successfully synthesized via MCP reaction. By tailoring the proportion of the two blocks, novel poly derivatives with satisfactory solution processability and film-forming ability can be readily achieved without complicated and high-cost post-processing film formation techniques. This cannot be realized by small PDI compounds or oligomers. The


Ring chromosome 6 (r(6)) is a rare disorder that mainly occurs as a ‘de novo’ event. Nonetheless, a wide phenotypic spectrum has been reported in r(6) cases, depending on breakpoints, size of involved region, copy number alterations and mosaicism of cells with r(6) and/or monosomy 6 due to loss of r(6).An 11-year-old male was referred with developmental delay, intellectual disability and microcephaly. Physical examination revealed additionally short stature and multiple facial dysmorphisms. Banding cytogenetic studies revealed a karyotype of mos 46,XY,r(6)(p25.3q27)[54]/45,XY,-6[13]/46,XY,r(6)(::p25.3→q27::p25.3→q27::)[13]/46,XY[6]/47,XY,r(6)(p25.3q27)×2[2]dn. Additionally, molecular karyotyping and molecular cytogenetics confirmed the breakpoints and characterized a 1.3 Mb contiguous duplication at 6p25.3.TBP gene in 6q27~qter, is likely to be the underlying cause of the phenotype. The phenotypic correlation and clinical severity in r(6) cases continue to remain widely diverse in spite of numerous reports of genomic variations.The present study has accurately identified copy number alterations caused by ring chromosome formation. A review of the literature suggests that hemizygous expression of  Ring chromosome 6 (r(6)) is an exceedingly rare disorder, first described in 1973 by Moore et al. . FormatiEven though being rare, a wide spectrum of phenotypic variability is observed in all cases of autosomal ring chromosomes. Congenital heart defects, intellectual disability, microcephaly, facial dysmorphism, failure to thrive and various abnormalities in the ocular, auditory and central nervous systems are frequently detected .In this study, a mosaic r(6) in a clinically abnormal, 11-year-old male was characterized in detail and compared with cases from the literature to share some new insights into the genotype-phenotype correlations.An 11-year-old male was referred for further diagnostics due to developmental delay, intellectual disability and microcephaly. He was the first child born to apparently healthy non-consanguineous parents. The mother had an uneventful pregnancy with no history of prenatal exposure to alcohol, drug or tobacco. Though his siblings (7-year brother and 3 years old sister) were phenotypically normal, his paternal cousin-sister was microcephalic and mentally challenged. No investigations were carried out in the affected cousin-sister.The proband was born by normal vaginal delivery. The birth weight was 1.5 kg and head circumference was 33 cm. Apgar score at birth was within the normal range. The patient was sitting without support at around 1.5 years. He could stand with support by 2 years and independent walking at 2.5 years. His speech development was delayed. He was not able to speak sentences and could not achieve proper bowel and bladder control even at the time of presentation (11 years). His height and weight were 108 cm and 14.5 kg respectively (below 10th centile); head circumference  was 42 cm (below 3rd centile). The proband portrayed short stature and microcephaly with developmental delay. The facial dysmorphism showed long face, small chin, large protruding ears, slightly upturned eyes, sparse eyebrows, large bulbous nose and thin upper lip Fig.  and b. SSample collection and written informed consent was obtained according to the need of the institutional ethics committee in accordance with Helsinki declaration. Chromosome analysis of the patient was performed with 72 h lymphocyte culture and standard GTG-banding. The karyotype was interpreted according to the International System for Human Cytogenetic Nomenclature (ISCN 2016)  as mos 4Karyotype of the parents was normal, confirming a ‘de novo’ origin of the r(6). Further analysis was carried out by molecular karyotyping using Affymetrix CytoScan™ 750 K array. Data was analyzed using Chromosome Analysis Suite (ChAS) and revealed: arr [GRCh37] 6pterp25.3156,974_665,234)x1,6p25.3x3,6q27qter×1. In other words, there was a partial terminal monosomy of overall 508 kb in 6p25.3 followed by a 1.3 Mb contiguous duplication in 6p25.3 and 448 kb terminal deletion in 6q27  syndrome frequently present with growth retardation, facial dysmorphism and microcephaly. The additional clinical features of the present case can most likely be attributed to the cryptic duplication event in the r(6).Major features associated either with 6p or 6q terminal deletions include defects of the anterior eye-chamber development, hearing loss, heart malformations, hypertelorism, mid-face hypoplasia, low set ears, developmental delay, intellectual disability, hypotonia, seizures, facial dysmorphism, and short neck , 11. Moshttps://decipher.sanger.ac.uk/).To establish genotype/phenotype correlations for r6), we compared the clinical features of the case under report to previously reported cases of r(6) Table ; Fig. 6 , we compDUSP22, IRF4, EXOC2, HUS1B; IRF4 (*601900) being the only OMIM morbid gene. Interferon regulatory factor 4 (IRF4) is a transcription factor essential for the development of T helper 2 (Th2), Th17 and Th9 cells, whose allelic variant influences variation in skin/hair/eye pigmentation 8. Dysregulation of IRF4 is associated with lymphoid malignancies [The deleted 508 kb subtelomeric region from 6pter to 6p25.3 contains 4 annotated genes: gnancies .EXOC2, LOC285768, FOXQ1, FOXF2, FOXC1, GMDS, FOXCUT; FOXC1 being the only morbid gene amongst them. Mutations in Forkhead box C1;  has been associated with phenotypes like Anterior segment dysgenesis 3 and Axenfeld Reigen syndrome type 3. Nishimura et al. [FOXC1 gene and ocular developmental abnormalities and glaucoma. Ophthalmologic examination in the patient under report did not reveal any abnormality and possibly point towards a less likely role of an extra copy of FOXC1 alone in the ophthalmological manifestations in such patients.The duplicated segment of 1.3 Mb at 6p25.3 was larger in size and contained more relevant OMIM genes-three copies each of a et al.  in theira et al.  reportedFOXC1 gene have also been speculated to be implicated in cardiac abnormalities [Apart from ocular manifestations, mutations and haploinsufficiency of malities , 32. Howmalities .LOC154449, DLL1, FAM120B, MIR4644, PSMB1, TBP, PDCD2 OMIM annotated genes. Amongst them, TATA-binding protein (TBP), a general transcription factor associates with aggregates in several polyglutamine disorders; is the only gene with a pathogenic potential [TBP as a potential candidate gene responsible for the phenotype of patients with subtelomeric 6q deletions, irrespective of the size of terminal deletion [C6orf70) gene in 1/14 (7.1%) patient with periventricular nodular heterotopia, developmental delay and epilepsy through whole exome sequencing. In addition, the authors silenced C6orf70 and two additional genes (phf10 and Dll1) in the developing rat neocortex and suggested that C6orf70 plays a major role in the control of neuronal migration and its haploinsufficiency or mutation causes periventricular heterotropia.A terminal deletion (448 kb) was detected (from 6q27 to 6qter) that encompass otential . A reasodeletion , 36. Mordeletion  has repoTBP might be implicated in the causation of the phenotype. Apart from these, the developmental delay, growth stunting and mild facial dysmorphism observed in the proband can also be attributed partially to the mitotic instability of the ring chromosome as the source of continuous production of secondary aneuploid cells [Developmental delay, intellectual disability, dysmorphism, seizures, dimpling of elbows and knees are some of the main features noted in such patients. Most of the above mentioned clinical features were also seen in our patient, raising a strong possibility pointing towards the hemizygous expression of id cells , 25. The