Ipomoea batatas leaf powders and evaluating their antihyperlipidemic effect on rats receiving a high-fat diet. Ipomoea batatas leaves were collected from four agroecological areas of Cameroon, and powders were obtained after washing, drying, grinding, and sieving. Standard analytical methods were used to determine the phytochemical composition of two varieties (IRAD-tib1yellow-V1 and IRAD-1112white-V2) from North Z1, Adamawa Z2, West Z3, Center Z4. The effect of I. batatas leaf powder on lipid metabolism was assessed in vivo by feeding different groups of rats with a high-fat diet supplemented with 5 and 10% of I. batatas leaf powder during 30 days. At the end of the experimentation, total cholesterols, triglycerides, LDL- (low-density lipoprotein-) cholesterol, HDL- (High-density lipoprotein-) cholesterol, ASAT (aspartate aminotransferase), ALAT , and creatinine were measured using commercial enzymatic kits . The results of phytochemical analysis of I. batatas leaf powders revealed that the total phenol content ranged from 660.173 mg GAE/100 gDW (Z1V2) to 657.76 mg GAE/100 gDW (Z3V2), the flavonoids content ranged from 282.25 mgEC/100 gDW (Z3V1) to 325.05 mgEC/100 gDW (Z4V2), and the anthraquinone content ranged from 324.05 mg/100 gDW (Z3V2) to 326.72 mg/100 gDW (Z4V2). The total antioxidant capacity ranged from 19.00 (Z1V1) to 23.48 mg AAE/gDW (Z3V2), while the IC50 ranged from 1.58 mg/mL (Z1V1) to 3.08 mg/mL (Z3V2). Rats fed a high-fat diet and supplemented with 5 and 10% of I. batatas leaf powder showed a significant (p < 0.05) reduction in body weight compared to the control with a reduction rate ranging from 6 to 10%. The consumption of I. batatas leaf powder for 30 days significantly (p < 0.05) reduced serum total cholesterol, LDL-cholesterol, triglycerides, ALAT, and creatinine level. These results suggest the use of I. batatas leaves as a phytomedicine in the treatment of cardiovascular diseases.The present study consists of analyzing the phytochemical composition of  Ipomea batatas (sweet potato) are an essential part of the human diet, providing the body with vitamins, minerals, proteins, some hormone precursors, and a high content of phytochemicals endowed with beneficial effects on health [I. batatas have been reported to be an excellent source of anthocyanins and many other bioactive compounds [I. batatas have focused on its antioxidant capacity due to its high content of flavonoids, β carotene, anthocyanins, and phenols [I. batatas leaves are used in traditional medicine . In addition to their antidiabetic and antiviral properties, Ipomoea batatas leaves have been used to treat infectious diseases in Brazil [I. batatas leaves has also been considered for the management of hyperlipidemia. Hyperlipidemia is a physiological disturbance characterized by an abnormal increase in cholesterol levels or blood lipoproteins [I. batatas [Ipomoea batatas leaves collected in different agroecological zones of Cameroon and to study their antihyperlipidemic activity in rats fed a high-fat diet.Nowadays, dietary habits have changed and the tendency is much more oriented towards traditional foods based on vegetables very rich in antioxidants to prevent cardiovascular diseases . Among vn health . In Japaompounds . In Braz phenols , 5. Thern Brazil . Phytochproteins . It is tproteins , 9. On tproteins . Many liproteins . A numbe batatas , 12. SomI. batatas IRAD-Tib1(V1) and white I. batatas IRAD-1112 (V2), were collected to approximately 20 cm starting from the apical pole in four areas of study: Sudano-Sahellian , Guinea savannah high zone , Western highland zone , and the rain forest with two modes  and were identified by Dr. Nérée Onguéné, plant taxonomist at the Agricultural Research Institute for Development of Yaounde , and Mr. MBONOMO Jean Moise, Technician of Institute of Agronomic Research for Development .Fresh leaves of two (2) varieties, yellow μm to produce fine leaf powder. The powders were stored in plastic packaging and hermetically sealed for further analysis.Leaves were washed and dried in the oven at 45°C for 48 hours and crushed with a moulin through a sieve mesh of 500 The Wolff  method wThe method described by Hsu  was usedμl of the sample (stock solution) was mixed with 125 μl of vanillin reagent (4% in methanol) and 60 μl of concentrated hydrochloric acid (35%). Absorbance was measured at 500 nm after 15 min incubation in the dark and at room temperature. The calibration curve was drawn with catechin (0-1 mg/mL). The proanthocyanidin content was expressed in mg catechin equivalent/g dry weight (mg CE/g DW).The vanillin method described by Broadhurst and Jones  was usedFor the determination of the anthraquinone content, the stock solution was evaporated under vacuum using a rotary evaporator to obtain the dry extract. Then, 1 g of extract was mixed with 30 mL of distilled water and boiled for 15 min under a reflux condenser. The analysis procedure was following with the steps described in the protocol of Sakulpanich and Gritsanapan , and the50  and were calculated using the exponential regression equation [The antioxidant activity of the samples was evaluated using three tests: the DPPH test , the FRAP (Ferric Reducing Antioxidant Power) test, and the TAC  test. The analysis procedures were following the steps described in our previous publication . For theequation .in vitro study. Before the study, the rats were weighed, divided into 6 groups of 6 rats, housed in a single cage, and acclimatized for one week with free access to tap water and food. The animals were kept in an environment of 25 ± 2°C with a relative humidity of 55 ± 5% under a cycle of 12 h light and 12 h dark. Procedures used in the study were approved by the Animal Ethics Committee of the Laboratory of Nutrition of the University of Yaounde I. The trial was registered with reference number: No. FW-IRB00001954.36 male Wistar albino rats of around 3 months, weighing around 250 g from the Laboratory of Nutrition, Faculty of Sciences, University of Yaounde I, Cameroon, were used for the ad libitum standard or normal diet. The positive control high-fat diet (TP-HFD) group received ad libitum high-fat diet. The TP-HFD-Z2V2 5% group, the TP-HFD-Z2V2 10% group, the TP-HFD-Z3V2 5% group, and the TP-HFD-Z3V2 10% group, fed ad libitum with a high-fat diet supplemented with doses of 5 and 10% (w/w) of I. batatas leaf powder from Zone 2/Varity 2 and Zone 3/Variety 2, respectively. The dose of dietary herbal powder supplementation was fixed on the basis of previous studies found in the literature [The standard and high-fat diets used to feed animals were formulated as proposed by Murase et al.  with sliterature , 21. TheThe levels of total cholesterol, HDL-cholesterol, and LDL-cholesterol in serum were measured using commercial enzymatic kits . TriglycHepatic function was assessed by the determination of Alanine Aminotransferase (ALAT) and Aspartate Aminotransferase (ASAT) activities using the spectrophotometer based on the protocols of commercial kits . The resThe creatinine content was determined using a spectrophotometer working at 520 nm with the Human Su-Crea 10052 kit , to knowp < 0.05. Regression analysis was performed using SigmaPlot 12 version 12.5.0.38 .Analyses were carried out in triplicate and data were subjected to analysis of variance to test the significance between treatments. Additionally, differences between means were tested using the Duncan Multiple Range test using Statgraphics Centurion XVI Software version 16.1.18 , and the values were considered statistically different at a risk p < 0.05) difference was observed between the samples, regardless of the origin of the harvest or the variety studied. The results obtained in this study are similar to those of Sun et al. [Fiber is the nondigestible polysaccharide residues of plants, and means of total fiber ranged from 8.15 ± 0.02 g/100 gDW (sample Z4V1) to 12.42 ± 0.09 g/100 gDW (sample Z1V2) . A signin et al. . They fon et al. , sweet pIpomea batatas leaf powder collected from the four localities. As can be seen in this table, the levels of total phenol, proanthocyanidins, and anthraquinones are very similar, regardless of the variety or origin of the plant. However, significant differences (p < 0.05) were observed for total phenol and proanthocyanidin contents. The total phenol content varies from 657.76 ± 0.31 gGAE/100 gDW to 660.10 ± 1.00 gGAE/100 gDW, and the proanthocyanidins content varies from 175.27 ± 0.05 gGAE/100 gDW to 176.04 ± 0.06 mgCE/100 g DW. The highest total phenol content was recorded in the Z4V2 sample, while the lowest value was recorded in the Z3V1 sample. On the other hand, the highest proanthocyanidin content was recorded in sample Z1V2, while the lowest value was recorded in sample Z2V1. No significant differences (p > 0.05) were observed in the anthraquinone content of the different sweet potato leaf samples. The average levels ranged from 324.05 ± 2.00 to 326.72 ± 2.52 mg/100 gDW. For the total flavonoid content, the variety of the plant has a significant influence on the values recorded in the sweet potato leaves since the lowest contents (282.25 ± 0.12 to 289.13 ± 0.96 mgCE/100 gDW) were observed in variety 1, while the highest contents (323.22 ± 0.20 to 325.05 ± 0.00 mgCE/100 gDW) were observed in varieties 2. Phytochemical compounds such as total phenols and flavonoids have also been detected in the leaves of harvested sweet potatoes in Ethiopia [Ethiopia . The totEthiopia . These aEthiopia . Total p50. Like the FRAP test, the antiradical capacity of these samples follows the same pattern, as no significant differences were observed for variety 1 . In addition, the highest antiradical activity was recorded for the Z3V2 sample with the lowest IC50 value (1.58 ± 0.02 mg/mL). The phenolic compound is considered as the major contributor to the total antioxidant capacity [I. batatas leaf powder could explain their use in the traditional treatment of some diseases. Further, considering their antioxidant capacity, Z2V2 and Z3V2 samples could be used to manage lipid profile such as lowering the effect on LDL-cholesterol and increase HDL-cholesterol levels.The antioxidant activity of the different samples was compared after estimation of the total antioxidant capacity (TAC) and Ferric reducing antioxidant power (FRAP) values in AAE/g DW, which are presented in capacity . The higIpomea batatas leaf powders according to similarities or differences in their bioactive compounds and their antioxidant activities. Additionally, the correlations between all measured parameters were revealed after performing the principal component analysis. From Principal component analysis (PCA) was carried out to group I. batatas leaf powder supplemented HFD (I. batatas leaf powder group (TP-HFD-Z2V2 5% and TP-HFD-Z3V2 5%) compared to the TP-HFD group feeding only with a high-fat diet (I. batatas leaf powder group (TP-HFD-Z2V2 5% and TP-HFD-Z3V2 5%) was similar, and no significant difference was observed between both . For the TP-HFD-10% group, a significant decrease in body weight (weight loss) was observed throughout the experimental period, and the final weight loss recorded was 2.9 and 3.2% of the initial weight, for TP-HFD-Z2V2 10% and TP-HFD-Z3V2 10%, respectively. It has been recommended that weight reduction programs should focus on achieving a modest weight loss of 7–10% of the initial weight. Therefore, I. batatas leaf powder could be used in weight reduction programs. The weight loss can be attributed to dietary fiber contains which were found in high amount in I. batatas leaf powder [I. batatas leaf powder collected in Japan. They found that 5% of the I. batatas leaf powder group showed significant suppression in final weight (390.6 ± 15.69 g) and adipose tissue weight (6.25 ± 0.95 g) compared to the positive control group feeding only a high-fat diet, and for which, the final weight and adipose tissue weight were 425.0 ± 24.97 g and 8.85 ± 1.32 g, respectively.The ingestion of the high-fat diet resulted in a gradual increase in body weight with time, whereas weight gain was suppressed in a dose-dependent manner in all groups fed the nted HFD . A signifat diet . At the f powder . In prevf powder , 35, evif powder , it is pf powder  who condp < 0.05) lower than those of rats fed the normal diet TN-STD (64.40 ± 0.32 mg/dL), while the opposite is observed for the final values of serum total cholesterol, triglycerides, and LDL-cholesterol concentration between these two groups of rats. For rats in the TP-HFD group, the final values obtained are 190.22 ± 0.94 mg/dL, 154.56 ± 0.28 mg/dL, and 111.67 ± 0.86 mg/dL, for serum total cholesterol, triglycerides, and LDL cholesterol, respectively, while for rats in the TN-STD group, the final values obtained are 166.35 ± 1.00 mg/dL, 118.92 ± 0.28 mg/dL, and 97.33 ± 0.54 mg/dL, respectively. Numerous studies have shown that increased levels of serum total cholesterol, triglycerides, and LDL-cholesterol increase the risk of developing atherosclerosis and coronary heart disease [I. batatas leaf powder was incorporated into the high-fat diet, significant reductions in serum total cholesterol, triglyceride, and LDL-cholesterol values were observed compared to the TP-HFD group. For rats in the TP-HFD-Z2V2-5% group, the final values obtained are 160.03 ± 0.36 mg/dL, 130.42 ± 0.33 mg/dL, and 90.17 ± 1.00 mg/dL, respectively. For rats in the HFD-Z2V2-10% group, the final values obtained are 153.15 ± 1.29 mg/dL, 122.98 ± 0.82 mg/dL, and 86.33 ± 1.15 mg/dL, respectively. These values were significantly lower than those of the TP-HFD-Z2V2 5% group. In addition, the supplementation with I. batatas leaf powder resulted in a significant increase in HDL-cholesterol in rats in the TP-HFD-Z2V2-5% and TP-HFD-Z2V2-10% groups  compared to the TP-HFD group. The decrease in LDL-cholesterol and the increase in serum HDL cholesterol levels contribute to an antiatherogenic condition [I. batatas, harvested in zone 3 and belonging to variety 2. The reduction in total serum cholesterol could be associated with the high dietary fiber content of I. batatas leaves. Several studies have shown that propionate generated from dietary fiber has hypocholesterolemic effects and compensates for the hypercholesterolemic effects of acetate production, which tends to increase total serum cholesterol via a mechanism probably involving hepatic lipogenesis [I. batatas leaf powder. However, they found that, the plasma triglycerides level in the 5% I. batatas group was significantly lower (66.96 ± 35.29 mg/dL) and approximately 38% of that in the positive control group (178.63 ± 44.76 mg/dL). On the other hand, other research studies have reported similar results to the present study as they found that the aqueous extract of I. batatas leaves collected in Cameroon restored blood lipid levels to normal values in the group of rats fed a high-fat diet [I. batatas leaves have hypolipidemic and antiatherosclerotic properties that justify their use in traditional medicine.The total cholesterol, LDL-cholesterol, HDL-cholesterol, and total triglyceride concentrations (mg/dL) of the different groups of experimental rats are shown in  disease , 37. Howogenesis , 39. Sevogenesis –46. For ogenesis –50. Theyogenesis . In contogenesis  found nofat diet . These aI. batatas leaf powder (TP-HFD-Z2V2-10% group and TP-HFD-Z3V2-10% group), no significant differences were found with an atherogenic index ranging from 0.25 ± 0.00 to 0.25 ± 0.01, respectively. However, these values were significantly low compared to the negative control groups and were also significantly low compared to rats fed a high-fat diet (TP-HFD) and to rats fed a high-fat diet and supplemented with 5% of I. batatas leaf powder . These results indicate that I. batatas leaf powder contains certain biological compounds that prevent the deposition of cholesterol or lipids in certain organs  and arteries. However, in general, at the end of the experiment (4 weeks of feeding), all the different groups of rats can be classified according to the atherogenic index values obtained, as high-risk groups for cardiovascular disease with an atherogenic index greater than 0.24.The TG/HDL-cholesterol ratio is linked to a risk factor for coronary heart disease, and the risk increases as this ratio also increase. This risk of cardiovascular disease is low when TG/HDL-cholesterol ratio is between −0.3 and 0.1, medium when TG/HDL-cholesterol ratio is between 0.1 and 0.24, and high when TG/HDL-cholesterol ratio is more than 0.24, as reported by Bo et al. [μmol/L, respectively. While the TN-STD group has an ALAT, ASAT values, and creatinine concentration of 16.06 ± 0.83 UI/L, 33.20 ± 0.59 UI/L, and 12.06 ± 0.78 μmol/L, respectively. Compared to the TP-HFD group, supplementation with I. batatas leaf powder significantly reduced the ALAT value in rats in the TP-HFD-Z2V2 and TP-HFD-Z3V2 groups. ALAT values ranged from 15.44 ± 0.34 UI/L to 26.28 ± 0.87 UI/L. Groups TP-HFD-Z2V2-10% and TP-HFD-Z3V2-10% had the lowest values , which were not significantly different from the TN-STD group (16.06 ± 0.83 UI/L). However, compared to the TN-STD group (33.20 ± 0.59 UI/L), the ASAT values (38.25 ± 0.73 UI/L to 41.37 ± 0.55 UI/L) remained significantly higher in all groups receiving a hyperlipidemic diet supplemented with I. batatas leaf powder. No significant difference was observed for TP-HFD-Z3V2-10% (38.27 ± 0.93 UI/L) and TP-HFD-Z2V2-10% (38.25 ± 0.73 UI/L) groups compared to the TP-HFD group (37.83 ± 0.43 UI/L). Regarding creatinine levels, the groups of rats fed a high-fat diet and supplemented with 5 and 10% of I. batatas leaf powder had a significantly lower creatinine level (9.73 ± 0.30 μmol/L to 11.19 ± 0.34 μmol/L) than the TN-STD (12.06 ± 0.78 μmol/L) and TP-HFD (12.70 ± 0.47 μmol/L) groups. These results show that supplementation of a diet with 10% of I. batatas leaf powder reduces the degree of liver damage and normalizes serum creatinine levels in rats fed a high-fat diet, indicating its protective effect on renal glomerular filtration capacity, as reported by Suanarunsawat and Songsak [The aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) values in the liver were quantified to determine the degree of liver damage in experimental rats and the results are shown in  Songsak .Ipomea batatas leaf powders, which also contain phytochemical compounds such as flavonoids, proanthocyanidins, and anthraquinones. I. batatas leaf powders also showed good antiradical and reducing activity, particularly samples Z3V2 and Z2V2 of variety 2 collected, respectively, in West and Adamaoua. In rats fed with a high-fat diet, supplementation with 10% I. batatas leaf powder was effective in reducing the levels of triglycerides, total cholesterol, and LDL-cholesterol as well as ALAT activity and creatinine concentration. The results of this study suggest the use of I. batatas leaves as a phytomedicine in the treatment of cardiovascular diseases. In the further development of this study, it would be interesting to identify, using analytical techniques, the phytochemical compounds endowed with these properties.This study reveals the high fiber content in


In this work, we first introduced 2-amino-3-hydroxypyridine and sodium borohydride as the nitrogen source and boron source to fabricate boron and nitrogen co-doped carbon quantum dots  with high fluorescence quantum yield (21.2%), which were synthesized through a simple, low-consumption and pollution-free one-pot hydrothermal method. The obtained N,B-CQDs are able to detect Cd2+ rapidly and sensitively through fluorescence enhancement, which may be ascribed to chelation enhanced fluorescence that is induced by the formation of the N,B-CQDs/Cd2+ complex. Simultaneously, N,B-CQDs can be used to detect l-cysteine because significant fluorescence quenching was observed when l-Cys was added into the N,B-CQDs/Cd2+ system. In the two fluorescence “turn-on” and “turn-off” processes, this fluorescent probe obtained a good linear relationship over Cd2+ concentration ranging from 2.5 µM to 22.5 µM with a detection limit of 0.45 µM, while the concentration of l-cysteine showed a linear relationship in the range of 2.5–17.5 µM with a detection limit of 0.28 µM. The sensor has been successfully used to detect Cd2+ and l-cysteine in real samples with satisfying results.Cadmium ions (Cd 2+ and l-Cys detection.Schematic presentation of the synthesis of N,B-CQDs and their application as an “on–off” fluorescent probe for Cd Among those various heavy metal ions, cadmium ions .16 However, the majority of the probes described above are usually either toxic or poor selective.In recent years, fluorescence method has become a popular detection method due to its advantages of simplicity, economy, high sensitivity and fast response speed. A variety of fluorescent probes have been researched, such as organic fluorophore molecules,17,18 In addition, carbon quantum points have been widely used in the fields of biological imaging,19,20 catalysis21 and sensors.22 In the above applications, it has been demonstrated that not merely surface functionalization/passivation, but also heteroatomic doping can improve the performance of CQDs. Boron (B), nitrogen (N), sulfur (S) and phosphorus (P) are the most common heteroatomic doping in carbon-based materials. The adjacent elements boron and nitrogen of carbon (C) in the periodic table have atomic radii similar to C, which makes it possible to effectively alter electronic characteristics and modulate physicochemical properties of carbon-based materials after doping. There are some previous researches have been reported to synthesize B and N co-doped carbon quantum dots based materials.23–26 However, the strategy for innovative and brief ways to make new B and N co-doped CQDs remains challenging.After more than a decade of development, carbon quantum dots (CQDs) have become a quite promising fluorescent probe with many unique advantages over traditional organic fluorophore molecules and semiconductor quantum dots, including excellent optical properties, favourable water solubility, low toxicity, excellent biocompatibility, and excellent sensitivity and selectivity.2+ and l-Cys rapidly and sensitively as an “on–off” fluorescent probe. When Cd2+ was added into the N,B-CQDs solution, the fluorescence of N,B-CQDs was sharply increased (turn on),which is attributed to the chelation enhanced fluorescence through the coordination reaction of surface functional groups with Cd2+. However, when l-Cys was introduced into N,B-CQDs/Cd2+ system, the fluorescence of the system was greatly quenched (turn off). This “on–off” fluorescence probe is selective and sensitive to Cd2+ and l-Cys with the detection limit of 0.45 µM and 0.28 µM, respectively. To our knowledge, this is the first paper to synthesize nitrogen–boron co-doped carbon quantum dots using 2-aminopyridine and sodium borohydride as N and B sources to detect Cd2+ based on fluorescence enhancement with “on–off” effect. Surprisingly, the probe is also suitable for Cd2+ and l-Cys analysis in real samples.In this work, we first introduced 2-amino-3-hydroxypyridine and sodium borohydride as raw materials to fabricate boron and nitrogen co-doped carbon quantum dots  with high fluorescence quantum yield (21.2%),which were synthesized through a simple, low-consumption and pollution-free one-pot hydrothermal method . The obt2.2.12 (99%) were purchased from Macklin Biochemical Co., Ltd . l-Cysteine hydrochloride monohydrate (99%) was purchased from Aladdin Reagent Co., Ltd, China. Sodium borohydride (98%) was purchased from Aladdin Reagent Co., Ltd, China. 6 amino acids including l-Lysine , glycine , l-arginine , d-glutamic acid , l-histidine (l-His. 99%) and d-valine  were purchased from Shanghai Xianding Biological Technology Co., Ltd . The metal salts used, including ZnSO4·7H2O (99%), KCl (99.5%), NaCl (99.5%), AgNO3 (99.8%), BaCl2·2H2O (99.5%), MgCl2·6H2O (98%), MnSO4·H2O (99%), CaCl2 (98%), K2CrO7 (99.8%), CoCl2·6H2O (99%) and CrCl3·6H2O (99%) were of analytical reagent grade and were used as received. Ultrapure water from a Ultrapure Water System  was used throughout the whole experiments.Citric acid (99.5%), 2-amino-3-hydroxypyridine (97%) and CdClUV-Visible absorption spectrum were measured by using a A11665 UV-Vis spectrophotometer . Fourier transform infrared spectroscopy (FTIR) spectrum were performed on a IRAffinity-1 spectrophotometer . The fluorescence measurements were recorded using an LF-1804005 fluorescence spectrophotometer . The slit width was set at 5 nm for both excitation and emission. Transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) images were performed on JEM-3200FS . X-ray photoelectron spectroscopy (XPS) were measured by ESCALAB 250 XI .2.2The B,N-CQDs was synthesized by one step hydrothermal. In a typical synthesis, firstly, 0.84 g citric acid and 0.5 g 2-amino-3-hydroxypyridine were dissolved in 30 mL of ultrapure water. Secondly, 0.08 g sodium borohydride was added. Then, the mixture was transferred to a teflon-lined autoclave (40 mL) and heated at 180 °C for 150 min. After cooling to room temperature, the above aqueous solution was dialysed through dialysis bag (1000 Da) for 48 h to remove raw materials. After dialysis, the solution was filtered through 0.22 µm membrane filter to remove the precipitate and frozen drying. Finally, the purified N,B-CQDs were brown powder and prepared for subsequent use.2.32SO4) as a reference. In this paper, quinine sulfate and N,B-CQDs solutions were prepared, and all of them had absorbance less than 0.05 at 350 nm (the maximum excitation wavelength of the standard). The QY of the N,B-CQDs was evaluated by the following equation:The quantum yield (QY) of N,B-CQDs was calculated with quinine sulfate  and 100 µL of Cd2+ with different concentrations. Kept the total volume at 4 mL and the final concentration of N,B-CQDs was 2.5 µg mL−1. After reacting at room temperature for 1 min, the fluorescence spectra were recorded at the excitation wavelength of 360 nm. The sensing system of l-Cys were prepared by mixing 4700 µL of ultrapure water, 100 µL of N,B-CQDs (0.1 mg mL−1), 100 µL of Cd2+ (250 µM) and 100 µL of l-Cys with different concentrations. Then kept the mixed solution react for 1 min. The fluorescence spectra of the system were recorded in the same way mentioned above.The sensing system of Cd2.52+ in tap water and lake water samples. The trap water was filtered through a 0.22 µm microporous membrane. The lake water was filtered through a 0.22 µm microporous membrane and centrifuged at 8000 rpm for 10 min. Then the different concentrations of Cd2+ were added to the obtained water samples for three parallel experiments. Moreover, the practical application of the l-Cys determination in human urine was carried out. The human urine sample was centrifuged at 8000 rpm for 10 min firstly, after that, various concentrations of l-Cys in human urine sample was added in the N,B-CQDs/Cd2+ system.To investigate the practicality of the system, the N,B-CQDs sensing system was applied to the detection of Cd3.3.128The TEM image  showed t−1 was indicated the stretching vibration of the O–H/N–H bond. The peak at 1640 cm−1 was designated as the stretching vibration of the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O bond, which was due to the carboxyl in the surface of CQDs.29 The two peaks at 1561 cm−1 and 1425 cm−1 were related to the stretching vibrations of CC and CN bonds of aromatic skeleton. The peaks at 700–800 cm−1 were attributed to the bending vibration of aromatic ring hydrogen. All above proved that there was pyridine structure in the structure of N,B-CQDs.30 The peaks at 1302 cm−1 ascribed to the stretching vibration of B–O bond,31 suggesting the existences of B element in the CQDs framework. And the peak at 1076 cm−1 was attributed to the C–O stretching vibration of C–O–C bond.The surface groups and chemical bonds of N,B-CQDs can be explored by FTIR spectrum. As shown in C, C–O, C–N–C and O–CO. The O 1s spectrum  emission behavior of the N,B-CQDs under different excitation wavelengths was investigated. As can be seen from the fluorescence spectrum , the flu37,38 which illustrates the extraordinary fluorescent properties and good application prospect of the N,B-CQDs in our work. Then, the effects of temperature, UV light irradiation time, pH value, ionic strength on the fluorescence stability of N,B-CQDs were evaluated. It is clear that the fluorescence intensity of N,B-CQDs kept stable when the temperature increased from 10 to 50 °C  and different concentrations of NaCl (0.5–3 M)  of quinine sulfate as the reference, the PL QY of N,B-CQD was about 21.2% at the excitation wavelength of 350 nm. This QY of the N,B-CQDs prepared in our work is higher than those previous researches reported, Fig. S2a, which i3.32+ detection was carried out in the pH = 7 ultrapure water after incubating with N,B-CQDs for 1 min. For the sake of the selectivity of the N,B-CQDs for Cd2+, the PL intensity variations before and after adding 250 µM of metal ions, including Cd2+, Zn2+, K+, Na+, Ag+, Ba2+, Mg2+, Mn2+, Ca2+, Cr6+, Co2+ and Cr3+ to the N,B-CQDs solution were investigated separately. As shown in F/F0 − 1) of N,B-CQDs is dramatically increased only in the presence of Cd2+,and other metal ions caused only little effect on the fluorescence intensity of the N,B-CQDs. 2+. It is clearly indicated that there was no significant effect on the PL intensity of the N,B-CQDs in the presence of interference metal ions. However, when Cd2+ ions were added to the N,B-CQDs solutions, remarkable fluorescence enhancement was noticed. From these results we can infer the conclusion that the N,B-CQDs have an excellent selectivity in detection of Cd2+ ions.In order to obtain optimal reaction conditions, the sample pH value (a), testing medium (b) and incubation time (c) were optimized. As shown in Fig. S3,2+, the detection limit for Cd2+ detection was explored. As shown in the 2+ ions increased, implying that the PL intensity of the N,B-CQDs is very sensitive to Cd2+ ions. The 2+. As illustrated in the inset of 2+ in the range of 2.5–22.5 µM is observed. The linear equation was F/F0 − 1 = 0.1498 + 0.0263C2+Cd with a good linear correlation (R2 = 0.9943). The limit of detection (LOD) for Cd2+ based on a signal-to-noise ratio of 3 was approximately 0.45 µM, which was comparable to other fluorescent probes based on carbon materials , attributes to complex of the surface functional groups with metal ions.46 Studies have proposed that chelation between surface functional groups and metal ions can decrease internal charge transfer (ICT), improve photoinduced electron transfer, and enhance emission.47,48 In our work, the enhancement of fluorescence may be due to the decreased electron donor capacity of electron donor groups (amino and pyridine nitrogen) when N,B-CQDs chelate with Cd2+, thus quenching the ICT transition and improving the photoinduced electron transfer to N,B-CQDs/Cd2+ complex. As shown in the Fig. S4,2+ concentration. This means the possibility of SPEF mechanism can be ruled out. In order to verify the formation of N,B-CQDs/Cd2+ complex, several kinds of characterization of the coordination of N,B-CQDs with Cd2+ or without Cd2+ were carried out. As illustrated in 2+, and the new peak at 1612 cm−1 demonstrated the formation of N,B-CQDs/Cd2+ complex. Furthermore, The zeta-potential increased from −1.69 eV to 3.26 eV after the addition of Cd2+  or plasma enhanced fluorescence (SPEF), of Cd2+ , which w3.52+ system used for l-Cys detection was investigated in the presence of l-Cys. As shown in 2+ system were investigated for comparison, only l-Cys exhibited notable quenching of the fluorescence. Thus, the system can be used for detection of l-Cys from different essential amino acids. As can be seen from l-Cys increasing, the fluorescence intensity of the system quenched gradually. This occurred because of that l-Cys have stronger binding preference toward Cd2+ than N,B-CQDs due to the formation of Cd2+–SR bond.49 The l-Cys. There are a good linear relationship with the concentration of l-Cys in the range of 2.5–17.5 µM. And the linear equation was 1 − F/F0 = 0.0326Cl-Cys − 0.0329 with a good linear correlation (R2) of 0.9921. The LOD for l-Cys based on a signal-to-noise ratio of 3 was approximately 0.28 µM, which was lower than those previous work using by “on–off” fluorescent probes  of three replicate detections for each sample below 5%. Moreover, the practical application of the l-Cys determination by N,B-CQDs/Cd2+ system in human urine was carried out. As shown in 2+ and l-Cys determination in real samples.In order to evaluate the practicality of this fluorescent probe, the N,B-CQDs sensing system was applied to detecting Cd4.2+ and l-Cys in aqueous solution as a “on–off” fluorescent probe. Delightedly, it has proved that the fluorescence enhancement mechanism of N,B-CQD detecting Cd2+ is realized by a chelation enhanced fluorescence generated by the coordination reaction between surface functional groups and Cd2+. Simultaneously, a obviously fluorescence quenching was observed when l-Cys was added into N,B-CQDs/Cd2+ system, which is due to the formation of Cd2+–SR bond that frees up N,B-CQDs. In the two fluorescence “turn-on” and “turn-off” processes, this fluorescent probe obtained a good linear relationship over Cd2+ concentration ranging from 2.5 µM to 22.5 µM with a detection limit of 0.45 µM, while the concentration of l-cysteine showed a linear relationship in the range of 2.5–17.5 µM with a detection limit of 0.28 µM. The sensor has been successfully used to detect Cd2+ and l-cysteine in real samples with satisfactory results. Furthermore, due to the fact that the synthesis method of N,B-CQD was straightforward, rapid, time-saving and cost-effective, it has provided a possibility for the practical application of Cd2+ detection with high sensitivity and selectivity in the future.In summary, we first introduced 2-amino-3-hydroxypyridine as nitrogen source to synthesize N,B-CQDs, and the quantum yield of N,B-CQDs reached up to 21.2%, which was higher than those in previous work. Based on the above characterization results, it can be seen directly that N atoms and B atoms were successfully doped into the structure of CQDs. N,B-CQDs was successfully used to detect CdThere are no conflicts to declare.RA-012-D1RA08219A-s001


The first step is a Suzuki–Miyaura coupling reaction between 2-bromophenylboronic acid and 1,4-diiodobenzene. The resulting 2,2′′-dibromo-1,1′:4′,1′′-terphenyl was reacted with tBuLi and hexamethylacetone to obtain the desired product. The crystal structure of H2[OO]tBu revealed an anti conformation of the [CPh2(OH)] fragments relative to the central phenyl. Furthermore, the hydroxyl groups point away from each other. Likely because of this anti–anti conformation, the attempts to synthesize first-row transition-metal complexes with H2[OO]tBu were not successful.A new sterically bulky chelating bis ligand 3,3′-bis, (H The ligand was synthesized in a two-step procedure as described in Fig. 2et al., 2010tBuLi followed by hexamethylacetone. The formation of the desired product H2[OO]tBu (35% isolated yield) was accompanied by the formation of significant amounts of p-terphenyl by-product (38% isolated yield). H2[OO]tBu was characterized by 1H and 13C NMR spectroscopy, high-resolution mass spectrometry, and X-ray crystallography. 1H NMR spectroscopy demonstrates the presence of two isomers at room temperature in an approximately 2:1 ratio, as manifested by two tert-butyl resonances (1.05 and 1.03 ppm) and two OH resonances (2.09 and 2.07 ppm). This observation suggests that, in contrast to H2[OO]Ph, various isomers of H2[OO]tBu do not readily interconvert at room temperature, possibly due to the more significant steric hindrance of the tert-butyl groups. An X-ray crystallography study (see below) suggests that in at least one of these isomers the hydroxyl groups are pointing away from each other; such an isomer is unlikely to coordinate a single metal in a chelating fashion. Accordingly, the reaction of H2[OO]tBu with several representative transition-metal amides  failed to produce isolable complexes.The success of this strategy led us to design a new, even bulkier ligand  disordered by symmetry over two positions. Selected bond distances, angles, and torsion angles appear in Table 12[OO]Ph, H2[OO]tBu manifests an anti conformation of the two ‘[CtBu2(OH)]’ donors relative to the central phenyl ring. In contrast to the structure of H2[OO]Ph, the hydroxyls point away from each other in the structure of H2[OO]tBu, leading to an overall anti–anti conformation. This disposition results in the placement of the tert-butyl groups above and below the central phenyl ring. The presence of bulky groups on both sides of the central phenyl is likely responsible for the distortion of the terphenyl fragment, which is indicated by the C10—C15—C16 angle of 130.70 (15)° and the C14—C15—C16 angle of 110.28 (15)°. Same distortion is likely responsible for the slight variation in  phenyl bond distances  was added dropwise and the resulting solution was stirred for 4 h. This reaction mixture was then transferred to a round-bottom flask containing hexamethylacetone  in 20 mL of hexane and stirred for 24 h. The organic contents were extracted using a dichloromethane–water solvent system. The organic phase was dried over MgSO4 and filtered. The filtrate was concentrated using a rotatory evaporator. The desired product H2[OO]tBu was separated in 35% yield  by column chromatography on silica gel using 3% ethyl acetate in hexane. para-Terphenyl  was found to be a major byproduct . Purified H2[OO]tBu was recrystallized from dichloromethane at 238 K to obtain colorless crystals suitable for X-ray crystallography. 1H NMR  δ 1.05  , 1.03 , 2.07 , 2.09 , 6.89 , 6.96 , 7.19 , 7.33 , 8.28 . 13C NMR  δ 29.99, 30.28, 65.61, 87.36, 125.15, 126.08, 129.47, 130.19, 130.92, 131.37, 134.40, 134.84, 140.76, 144.07, 146.32. HRMS (m/z): Calculated [M - H]+ 515.39, found 515.36.2,2′′-Dibromo-1,1′:4′,1′′-terphenyl (Velian 2[OO]tBu) over two positions. Two alternating positions were identified from the difference-Fourier maps and refined to 50% occupancy. The CH2Cl2 solvent was also disordered by symmetry over two positions and refined with 50% occupancy.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021013438/jy2014sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021013438/jy2014Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021013438/jy2014Isup3.cmlSupporting information file. DOI: 2129676CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Yet this complex conundrum can be alleviated and mitigated through sound soil and water management practices. Despite considerable literature on Conservation Agriculture (CA) as a strategy to improve the resilience and mitigation capacity of agroecosystems, there is still paucity of information on the impacts of CA on crop production and environmental quality on the Plateau. In order to fill this gap this study examined the effects of no-till and straw mulch on crop productivity and greenhouse gas fluxes in agroecosystems on the Plateau where farmers’ common practice of conventional tillage (CT) was tested against three CA practices: conventional tillage with straw mulch (CTS), no-till (NT) and no-till with straw mulch (NTS). The results indicated that all three CA practices  markedly increased soil water content (SWC), soil organic carbon (SOC) and soil total nitrogen (STN) but reduced soil temperature (ST). Average grain yields were 854.46 ± 76.51, 699.30 ± 133.52 and 908.18±38.64 kg ha-1 respectively under CTS, NT and NTS indicating an increase by approximately 33%, 9% and 41% respectively compared with CT (644.61 ± 76.98 kg ha−1). There were significant (p < 0.05) reductions of Net CO2 emissions under NT (7.37 ± 0.89 tCO2e ha−1y−1) and NTS (6.65 ± 0.73 tCO2e ha-1y-1) compared with CTS (10.65 ± 0.18 tCO2e ha−1y−1) and CT (11.14 ± 0.58 tCO2e ha−1y−1). All the treatments served as sinks of CH4but NTS had the highest absorption capacity (−0.27 ± 0.024 tCO2e ha−1y−1) and increased absorption significantly (p < 0.05) compared with CT (−0.21 ± 0.017 tCO2e ha−1y−1); however, CA did not reduce emissions of N2O. These had an influence on Global warming potential (GWP) as NT and NTS resulted in significant reduction in net GWP. Grain yield was significantly correlated positively with SOC and STN (p < 0.05); ecosystem respiration was also significantly correlated with SWC and ST while CH4 flux was highly correlated with ST (p < 0.001). Crop yield and GHG responses to CA were controlled by soil hydrothermal and nutrient changes, thus improving these conditions through adoption of sustainable soil moisture improvement practices such as no-till, straw mulch, green manuring, contour ploughing and terracing can improve crop resilience to climate change and reduce GHG emissions in arid and semi-arid regions.The effects of climate change such as dry spells, floods and erosion heavily impact agriculture especially smallholder systems on the Northwestern Loess Plateau of China. Nonetheless agriculture also contributes to global warming through the emission of greenhouse gases such as CO These gne (CH4) . Meanwhine (CH4)  while lone (CH4) . In all Wheat is a crop with global importance  and is c2  cycle. The practice of CT where mechanical means is employed in land preparation causes rapid soil organic matter decomposition and oxidation of soil C to CO2 . This ma2 , chemica2  and biol2 . Managem2  is key t2 , increas2  and incr2 . But the2 , reduced2  and no e2 . Differe2 . It is n2, CH4 and N2O fluxes as affected by conservation tillage and (3) to identify the mechanisms that control the responses of yield and greenhouse gases to tillage practices in dryland areas.Thus the objective of this study was (1) to examine the influence of no-till and straw mulching soil management practices on crop yield (2) to analyse the dynamics of CO−3. Average soil organic carbon (SOC) was 6.21 gkg−1 while total nitrogen content was 0.61 gkg−1. Precipitation, maximum and minimum temperatures for the experimental period are shown in This experiment was conducted for two years (2017-2018) in the Anjiapo catchment on the western Loess Plateau in Gansu province at the Soil and Water Conservation Research Institute in Dingxi . For this study we have continuous data of forty two years . The soil is formed from Loess with a sandy-loam texture, with average soil bulk density of 1.26 gcm2O5) at a rate of 146 kg/ha and urea (46%) at a rate of 63 kg/ha. Three rows per plot were harvested for determination of aboveground and below ground plant products at physiological maturity. Aboveground biomass was determined by oven drying of plants at 80 °C to constant weight , conventional tillage with straw mulch (CTS), no-till (NT) and no-till with straw mulch (NTS). Sowing was conducted in spring (mid-March) in both years while crops were harvested in late July to early August. In the tilled plots, soils were tilled at two different times by manual inversion with shovels to a depth of 20 cm; first in October of the previous year and again in March just before planting. Glyphosate (30%) herbicide was applied to control weeds in the plots. Wheat straw (dry weight of 3.75 ton/ha) was spread uniformly on all straw-treated plots immediately after planting. Chemical composition of the wheat straw is shown in t weight , while gt weight .2O software. Chamber temperature was recorded using a handheld digital thermometer . Soil moisture and soil temperature data were taken concurrently with gas sampling.Soil water content and soil temperature at 0–10 cm depth were measured using EM50 data logger and GS3 soil moisture, temperature and EC sensor . The data was sampled every 2 min and subsequently downloaded onto the computer using the ECHSoils were sampled at 0–10, 10–20 and 20–40 cm with a soil auger (4 cm diameter) for determination of soil organic carbon (SOC) and soil total nitrogen (STN). SOC was determined by the Walkley-Black dichromate oxidation method  while ST4 and CO2 analysis and Electron capture detector (ECD) for N2O analysis. The FID operates at a temperature of 250 °C, and H2 flow rate of 35 cm3 min−1. Peak areas of CO2, CH4 and N2O were analyzed in Echrom-ChemLab software. Before the analyses of sample gases, calibrations were done with standard gas obtained from Shanghai Jiliang Standard Reference Gases Co., Ltd, China. Concentrations of the standard gases were 456.00 ppmv for CO2, 2.00 ppmv for CH4 and 0.355 ppmv for N2O. The sample gas concentrations obtained for the five sampling times were plotted against time in order to obtain the change in concentration over the sampling time. CO2 emissions in terms of ecosystem respiration (Reco), CH4 and N2O fluxes were calculated as shown in the The gas sampling procedure was conducted between September, 2017 and January, 2019. The static dark chamber and Gas chromatography (GC) method as described by p < 0.05. Linear and non-linear regressions were used to examine the relationships between crop yields, soil properties and greenhouse gas fluxes. The exponential and power equations were used to describe the relationship between ecosystem respiration, soil temperature and soil water content as shown in equations 11 and 12 respectively . One-way Anova was conducted and treatment means were separated using the Duncan’s multiple range tests (DMRT) at ectively .Soil water content (SWC) was higher in NTS than all other treatments while CT had the lowest SWC at almost all sampling times . ConventSoil temperature, as shown in p < 0.05) under CTS, NT and NTS. For the 0–10 cm SOC values were 9.98 ± 0.73, 11.97 ± 0.5, 11.81 ± 0.09 and 12.57 ± 0.62 gkg−1 respectively for CT, CTS, NT and NTS. Compared with CT, SOC was increased by 19.95%, 18.38% and 26.03% respectively under CTS, NT and NTS within the 0–10 cm soil depth. A similar trend was observed within the 10–20 cm profile where SOC was also increased by 12.51%, 10.76% and 14.26% respectively under CTS, NT and NTS. However in the 20–40 cm depth there was a little deviation where SOC in NTS and CTS were significantly greater than CT but NT showed no significant difference. Meanwhile SOC decreased along soil depth irrespective of treatment.Conservation Agricultural practices increased SOC at all depths  and 3C. p <0.05) in STN variations among treatments in the 0–10 cm and the 10–20 cm depths , STN was increased significantly (p<0.05) by 21.59%, 43.75% and 65.91% respectively in CTS, NT and NTS. Similarly, within the 10–20 cm depth, STN was significantly (p<0.05) increased under CTS (0.20 ± 0.07 gkg−1), NT (0.24 ± 0.10 gkg−1) and NTS (0.31 ± 0.01 gkg−1) compared with CT (0.17 ± 0.01 gkg−1). Nonetheless there were no significant differences (p >0.05) in STN within the 20–40 cm depth. However there were observed reductions of STN along soil depth in all treatments.There was also significance  for the period (2017-2018) but grain yield was not significantly increased under NT. There were however slight interannual variations in yield response to treatments. Grain yields were generally higher in 2018 than in 2017. Also in 2017 NTS showed the highest grain yield but in 2018 CTS showed the highest grain yield. While only NTS significantly increased grain yield in 2017, in 2018 both NTS and CTS significantly improved grain yield.Tillage and straw treatments influenced grain yield in both years . Average2 than non-tilled soils. In the growing season, average CO2 emission rates were 270.475 ± 11.03, 262.88 ± 0.20, 183.83 ± 34.05 and 190.72 ± 19.20 mg C m−2 h−1 in CT, CTS, NT, and NTS respectively, resulting in emission reduction in CTS, NT, and NTS by 2.81%, 32.03% and 29.48% respectively. In the non-growing season, emissions were relatively lower at rates of 30.55 ± 1.71, 45.51 ± 3.88, 31.74 ± 1.35 and 34.15 ± 5.71 mg C m−2 h−1 respectively in CT, CTS, NT, and NTS.Ecosystem Respiration for all treatments are shown in 4  of CH4 and N2O, GWP and GHGI of all treatments are shown in The Net CO2 fluxes in NTS and NT (6.65 ± 0.73 and 7.37 ± 0.89 tCO2e ha−1y−1respectively) were significantly lower than those in CT and CTS (11.14 ± 0.58 and 10.65 ± 0.18 tCO2e ha−1y−1respectively), showing reduced net carbon exchange into the atmosphere under NTS and NT. Similarly, GWP was greater in CT than all other treatments with significant reductions in NT and NTS (p < 0.05). Compared with CT, the reduction in GWP was 2.83%, 33.40%, and 40.35% under CTS, NT and NTS respectively. The GHGI is a yield-scale quantification of GWP, thus is a factor of GWP and Grain yield. It shows the contribution of the cropping system to global warming per unit grain yield. Our results showed that CT was the highest contributor of global warming per unit grain produced compared with other treatments. Significant reductions of GHGI were found in NTS and NT (p < 0.05).Net COp < 0.05) of grain yield and SOC and STN (Grain yields were highly influenced by soil nutrients. There were significant positive correlations ( and STN  at the 0 and STN . However2 emission in the form of ecosystem respiration (Reco) increased exponentially as ST increased; ST-Reco relationship followed an exponential function and was highly significant (p < 0.001) and positive with R2 values of 0.68, 0.63, 0.80 and 0.80 respectively in CT, CTS, NT and NTS while SWC-Reco relationship was best described by a power function and also showed highly significant (p < 0.001) positive correlations with R2 values of 0.06, 0.10, 0.14 and 0.07 respectively in CT, CTS, NT and NTS. Similarly, for CH4 fluxes, ST-CH4 relationship was an exponential function and highly significant as well (p < 0.001) while SWC-CH4 relationship was best described by a linear function, albeit not significant. Meanwhile ST and SWC did not seem to explain variations of N2O as the correlations were not significant except in ST-N2O relationship under CTS.Greenhouse gas fluxes were generally influenced by ST and SWC as shown in Increased SWC and ST reduction in NTS  is in liOther studies have also reported increased SOC stocks after adoption of conservation tillage practices  which isHigher grain yield under CA practices  is in tap < 0.05) in NT and NTS compared with the tilled soils  utilizes H2/CO2 and acetate as methane producing precursors, and produces far higher methane than the methanogen at lower temperatures (Methanosaetaceae), which uses only acetate as methane producing precursor , No-till (NT) and no-till with straw (NTS)) to conventional tillage (CT). Our study showed that conservation tillage practices especially NTS improved soil water content and reduced soil temperature. Soil organic carbon and total nitrogen were also significantly improved under conservation practices especially within the top soil layer (0–20 cm). There was also significant improvement in average grain yield under NTS and CTS. Conservation tillage further reduced net CO10.7717/peerj.11064/supp-1File S1Click here for additional data file.10.7717/peerj.11064/supp-2File S2Click here for additional data file.


TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous   Optimized non-integrating alpharetrovirus-based Gag.MS2 chimeric particles were used to vectorize CRISPR-Cas9-based genome engineering tools to efficiently knockout target genes and optionally induce HDR in reporter and primary cells. One possibility is the use of non-integrating reverse transcription disabled retroviral vectors,38,SpCas9 mRNA and sgRNA transcripts.,SpCas9 mRNA and sgRNA resulted in the generation of potent and significantly less cytotoxic g.Gag.MS2.CRISPR-Cas9 all-in-one (g.Gag.MS2.CRISPR) particles, which induced efficient knockout of several genes in various cell types.Recently, we and others developed retrovirus-based MS2 bacteriophage (MS2) chimera for reversible delivery of the multi-component CRISPR-Cas9 technology into target cells.TP53/Trp53 genes in several primary cell sources. Noteworthy, co-packaging of SpCas9 mRNA and multiple sgRNA transcripts into a.Gag.MS2 chimera showed efficient, simultaneous, and targeted knockout of up to three genes. Co-transfection of a single-stranded DNA oligonucleotide (ssODN) during CRISPR-Cas9 particle production led to all-in-one particles, capable of homology-directed repair (HDR) in HT1080 and primary cell cultures. In summary, we report optimized a.Gag.MS2 particles as a versatile tool for short-term and dose-controlled delivery of CRISPR-Cas9 into target cells.In the present study, we aimed to further improve the potency of this approach and transferred the gammaretroviral MLV-based technology to the recently described alpharetroviral Rous sarcoma virus (RSV)-based vector platform.Cas9 and sgRNA transcripts into target cells. The preservation of most Gag subdomains should allow the generation of retrovirus-like particles, which protect the non-retroviral RNA cargo from extracellular nucleases and facilitate, together with the envelope glycoprotein, introduction of the RNA cargo into cells in a non-toxic manner.The recently described g.Gag.MS2.CRISPR particles were designed for spatiotemporal co-delivery of Streptococcus pyogenes Cas9 (SpCas9.TS) mRNA and RNA-Pol-III-driven sgRNA.TS transcripts, which contain the TS hairpins incorporated either within (TS.inc sgRNA) or adjacent (TS.adj sgRNA) to the sgRNA scaffold.,Tet methylcytosine dioxygenase 2 (Tet2) gene resulted in efficient knockout rates of a far-red fluorescent RFP657 reporter gene that contained the Tet2 recognition site downstream of its start codon  efficient than respective Tet2.TS.inc sgRNA particles when equal supernatant volumes were compared.To realize this concept, we designed a g.Gag.MS2 precursor protein variant, in which the NC was substituted by a genetically fused MS2CP dimer that was separated from the Gag capsid (CA) subdomain by the natural retroviral protease site A. In addThe recently designed g.Gag.MS2.CRISPR particles showed efficient and reversible delivery of CRISPR-Cas9 components into various target cells.As several retroviruses have co-evolved in different vertebrate hosts over millions of years,Tet2-targeting a.Gag.MS2.CRISPR (a.Gag.MS2.CRISPR.Tet2) particles carrying either Tet2.TS.inc or Tet2.TS.adj sgRNAs and transduced human HT1080-based RFP657.Tet2 reporter cells.,RFP657.Tet2 knockout rates compared with Tet2.TS.adj sgRNA-containing particles. Remarkably, the application of only 0.5 μL of a.Gag.MS2.CRISPR.Tet2.TS.inc supernatant yielded mean RFP657.Tet2 knockout rates of 46%. As this was 20-fold less supernatant volume than what was needed for comparable knockout rates mediated by Tet2.TS.adj-containing g.Gag.MS2.CRISPR particles  enhancer/promoter sequences , which represent a promising cell source for basic and clinical research.equences A.57,58 Iernatant D. Intereernatant B controlernatant A. In comernatant B.In summary, the transfer of the Gag.MS2-based technology from the gamma- to the alpharetroviral background significantly improved its efficiency and resulted in enhanced CRISPR-Cas9-mediated knockout rates in human fibroblasts as well as hiPSC. Furthermore, the non-integrating nature of Gag.MS2 particles seemed to be beneficial in hiPSC, as they outperformed their integrating LIT correlates in terms of (knockout) efficiency and cytotoxicity ratios.SpCas9.TS mRNA transcripts in respective supernatants . Conversely, g.Gag.MS2-based supernatants had 2.3-fold-more Tet2.TS sgRNA than a.Gag.MS2.CRISPR.Tet2 supernatants. Interestingly, in both types of supernatants, we detected SpCas9 protein, whose levels varied between supernatant batches  or Trp53 (murine), respectively, as altered expression of the p53 master regulator protein may promote cell survival and division, which would facilitate our experimental readout.,SpCas9.TS transcripts. Regarding absolute SpCas9.TS mRNA numbers, both supernatant types showed on average 8.3 × 106 (TP53) or 1.2 × 107 (Trp53) SpCas9.TS mRNA copies/μL.We next tested the applicability of our a.Gag.MS2.CRISPR particles in human and murine primary cells. We chose to knockout human or mouse endogenous tumor suppressor genes TP53 insertion and deletion (indel) frequencies within these cultures via tracking of indels by decomposition (TIDE) analysis.SpCas9.TS mRNA copies/cell already yielded in TP53 knockout rates of ∼13%, which could be further increased to ∼93% knockout with the highest particle dose used  and primary human umbilical-cord-blood-derived CD34+ hematopoietic stem and progenitor cells (hCD34+ HSPCs). Based on the results in NuFF cells, we applied particle doses up to 4,150 SpCas9.TS mRNA copies/cell. Remarkably, determined TP53 knockout rates in treated PHH cultures were as efficient as those that we measured in transduced NuFF cultures (TP53 knockout rates in hCD34+ HSPCs were moderate as we achieved indel rates ranging from 6% to 36% (SpCas9.TS mRNA copies/cell on 2 consecutive days and obtained mean Trp53 indel rates of 31% (CF1) and 25% (C3H).First, we transduced human newborn foreskin fibroblasts (NuFFs) with indicated increasing doses of a.Gag.MS2.CRISPR.TP53 supernatants and assessed the ose used A. To tesver time B. Comparcultures C. In con% to 36% D. Finall% to 36% E. As preEGFP is converted to enhanced blue fluorescent protein (EBFP) by the substitution of two amino acids (T66S and Y67H).EGFP, and no TS-carrying ssODN donor templates encoding the T66S and Y67H mutations, including flanking homology arms and with or without a stabilizing phosphorothioate (−PT or +PT) bond modification  of ∼1. Independent of the co-packaged −PT or +PT ssODN, transduction of EGFP-expressing HT1080 cells with a.Gag.MS2.CRISPR.HDR particles resulted in comparable and efficient EGFP knockout rates of up to ∼56% . However, the use of PT-stabilized ssODNs significantly increased HDR events and resulted in HDR rates of up to 5% already with 30 μL applied supernatant. To further confirm these results, we analyzed the performance of a.Gag.MS2.CRISPR.HDR supernatants in the presence of non-homologous end joining SpCas9.TS mRNA, EGFP.TS sgRNA, and ssODN content (SpCas9.TS mRNA (6.6 × 106 copies/μL) and ssODN template (3.6 × 106 copies/μL), while the EGFP.TS sgRNA transcript levels were on average 6.1-fold higher (3.1 × 107 copies/μL). Subsequently, we transduced HT1080 EGFP reporter cells with 30 μL  a.Gag.MS2.CRISPR.HDR supernatant in the absence (w/o) or presence of DMSO, inh.NHEJ, or inh.HDR  and MEFs (18%–31%). Here, it would be interesting to further test other particle doses and/or explore other transduction conditions in combination with small molecules that reversibly interfere with intrinsic cellular immunity.79Finally, we demonstrated efficient knockout of endogenous articles . While iSpCas9.TS mRNA and sgRNA.TS transcripts within particles, and differences in retrovirus biology.Taken together, the superior performance of a.Gag.MS2.CRISPR supernatants can most likely be explained by enhanced particle titers, more favorable stoichiometries of SpCas9.TS mRNA and ∼1,000–2,000 sgRNA.TS copies per cell. Comparing this to routinely used physicochemical CRISPR-RNA transfer protocols, which used ∼4 × 105–8 × 106SpCas9 mRNA copies/cell and ∼1 × 108–1 × 109 gRNA copies/cell,,,SpCas9.TS mRNA or 50,000- to 1,000,000-fold-less sgRNA.TS transcripts. These data illustrate that spatiotemporal co-delivery of CRISPR-Cas9 components by a.Gag.MS2.CRISPR particles is a cost-effective resource for efficient genome editing of target cells.To achieve ∼50% knockout efficiency in HT1080-based RFP657.Tet2 reporter cells and highly permissive PHHs and NuFFs, we used ∼500–1,000 As all features necessary for productive cellular entry are retained in a.Gag.MS2 particles, it is very likely that they enter the cell in the same manner as WT retroviruses. This evolved and sophisticated entry strategy not only mediates efficient cellular uptake of a.Gag.MS2 particles but also promotes their efficient endosomal release (and the incorporated RNA cargo) into the cytosol, which is currently the bottleneck of non-viral, particle-based delivery strategies.,in vitro and in vivo.,To further improve the a.Gag.MS2-based delivery method, particle-destabilizing agents and/or capsid mutations should be experimentally tested.The construction of the vectors used in this work is explained in detail in the “+ Jurkat cells6 cells/well and cultivated in William's medium E supplemented with 1 μM insulin, 1 μM dexamethasone/fortecortin, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 4 mM L-glutamine, and 5% FBS . Umbilical-cord-blood-derived hCD34+ HSPCs were obtained from the Department of Gynecology and Obstetrics at Hannover Medical School after written informed consent as approved by the Ethics Committtee of Hannover Medical School. Mononuclear cells were separated from diluted cord blood (1:2 with PBS) by means of density gradient centrifugation using Leucosep separation tubes  and Biocoll (Biochrom) as a separation medium. The isolation of the mononuclear cells was performed according to the manufacturer's instructions. Purification of CD34+ HSPC cells was done by magnetic cell separation using the CD34 MicroBead Kit  according to the manufacturer's protocol. The CD34+ HSPCs were cultured and expanded in serum-free StemSpan medium  supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ng/mL hSCF, 50 ng/mL hTPO, 100 ng/mL hFLT3 , 500 nM StemRegenin1 (STEMCELL Technologies), and 35 nM UM171 . The hiPSC.RFP657.Tet2 reporter cells are derived from the former described H2E6C hiPSC lineHuman embryonic kidney 293T (293T) cells and human fibroblast HT1080-derived reporter cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate . HT1080 RFP657.Tet2 and HT1080 EGFP reporter cells were additionally cultured in the presence of 1 μg/mL puromycin . Human CXCR46 cells were seeded per 10 cm culture dish . g.RIT.CRISPR.Tet2 vector particles were generated by co-transfection of 5 μg pSV40enh.RSF91.hU6.sgRNA.Tet2.SpCas9.P2A.EGFP.gPREViral supernatants were produced after transient transfection of 293T cells using standard calcium phosphate precipitation.Alpha- or gammaretroviral Gag.MS2.CRISPR particles were generated by transfecting each 2.5 μg of non-retroviral pC1.SpCas9.P2A.EGFP.TS.gPRE and respective sgRNA.TS expression plasmids together with 5 μg pcDNA3.MLV.CA.pr.MS2CP (g.Gag.MS2) or 3 μg pcDNA3.ASLVco.NC.pr.MS2CP (a.Gag.MS2) and 2 μg pMD2.G VSV-G expression plasmids. For the generation of a.Gag.MS2.CRISPR.HDR particles 0.43 nmol +PT ssODN or −PT ssODN templates were added to the transfection.g for 2 h or overnight at 13,238 × g at 4°C.In case of Gag.MS2 particle production, respective transfection efficiencies were assessed by flow cytometric analysis of 293T producer cells after the last supernatant harvest. All produced viral supernatants were concentrated 100-fold by ultracentrifugation at either 82,740 × 4 HT1080 RFP657.Tet2 reporter cells were seeded in a 12-well plate. For the transduction of Jurkat-based reporter cells, 1 × 105 cells were plated per well of a 24-well plate. Indicated amounts of viral supernatants were applied to the cells, and the transduction was assisted by 4 μg/mL protamine sulfate (Sigma Aldrich) and centrifugation at 400 × g and 37°C for 1 h. The transduction efficiency was determined via flow cytometry 5 to 6 days post-transduction. hiPSC-based RFP657.Tet2 reporter cells (5 × 104) were transduced with indicated amounts of the respective Gag.MS2.CRISPR.Tet2 supernatants or with LIT.CRISPR.Tet2 particles at an MOI of 2 and 5. Transduction was performed in single-cell suspension in the presence of 4 μg/mL protamine sulfate and 10 μM ROCK-inhibitor Y-27632  for 1 h at 37°C and 5% CO2. Every 10 min, the tubes were flicked to keep the cells in suspension. After the 1 h incubation time, the cells were seeded on Geltrex in conditioned hiPSC medium containing 10 μM ROCK inhibitor for the first 24 h. The medium exchange was accomplished the following day and every other day. Knockout rates were determined 5 days post-transduction via flow cytometry. Primary wt NuFF cells were seeded in a density of 5 × 104 cells/well in a 12-well plate the day before transduction. Transduction was performed with indicated SpCas9.TS mRNA vector doses, 8 μg/mL hexadimethrine bromide , and centrifugation at 400 × g and 37°C for 1 h. The PHHs were directly transduced after seeding (1.5 × 106 cells/well) with indicated SpCas9.TS mRNA vector doses in the presence of 8 μg/mL Polybrene. For transduction of hCD34+ HSPCs, 1 × 105 cells were plated per well of a 48-well plate and transduction was assisted by addition of 8 μg/mL Polybrene and 1 mg/mL Synperonic F 108 (Sigma-Aldrich). For HDR experiments, 5 × 104 HT1080 EGFP or NuFF EGFP reporter cells were seeded in a 12-well plate. One day later, the cells were transduced in the presence of 4 μg/mL protamine sulfate (HT1080 EGFP) or 8 μg/mL Polybrene (NuFF EGFP) with or without 1 μL/mL DMSO, 5 μM YU238259 , or 1 μM M3814  followed by centrifugation at 400 × g and 37°C for 1 h. HDR efficiency was determined 8 days post-transduction via flow cytometry.The day before transduction, 5 × 10SpCas9.TS mRNA, respective sgRNA.TS transcripts and/or +PT ssODN templates in Gag.MS2 supernatants, a quantitative real-time PCR was performed. In brief, g.Gag.MS2- and a.Gag.MS2-based particles were produced and 100-fold concentrated, and resulting pellets were resuspended in PBS with 20 mM HEPES. To eliminate potential plasmid contamination, supernatants were treated with 2 U TURBO DNase (Ambion/Thermo Fisher Scientific) for 1 h at 37°C. Subsequently, the RNA was extracted from particles using the QIAGEN RNeasy Micro Kit  following the manufacturer's protocol with (SpCas9.TS mRNA and sgRNA.TS) or without (+PT ssODN) an additional DNase digestion step on the column. To adjust for the potential loss of RNA transcripts or +PT ssODNs during the extraction procedure, DNase-treated supernatants were mixed with a fixed volume of no PRE-encoding RIT.Cre supernatant before RNA extraction. While the amount of +PT ssODNs was directly determined by quantitative real-time PCR after isolation (primers 5′-GGACTTGAAGAAGTCGTGCTG-3′ and 5′-ACCCTCGTGACCACACTG-3′), extracted RNA was reverse transcribed into cDNA prior to quantitative real-time PCR using the QuantiTect Reverse Transcription Kit (QIAGEN). Detection of SpCas9.TS cDNA was performed using the PRE-recognizing primer pair 5′-GAGGAGTTGTGGCCCGTGT-3′ and 5′-TGACAGGTGGTGGCAATGCC-3′. The reverse-transcribed sgRNA.TS variants were detected with a common reverse primer 5′-CAAGTTGATAACGGACTAGCCTT-3′ (binding to the sgRNA scaffold) together with a specific forward primer for the different protospacer sequences . Cre cDNA was amplified with primers 5′-AACCATTTGGGCCAGCTAAACA-3′ and 5′-AGAGCCTGTTTTGCACGTTCA-3′. Total amounts of SpCas9.TS mRNA, sgRNA.TS, Cre mRNA, and +PT ssODN copies were calculated with the help of serial dilutions of individual plasmid or ssODN standards, respectively. The signals for +PT ssODN, SpCas9.TS, or sgRNA.TS were corrected with signals obtained for Cre.To determine the content of The amount of SpCas9 protein in 100-fold concentrated Gag.MS2.CRISPR.Tet2 supernatants was determined via the EpiQuik CRISPR-Cas9 Assay ELISA Kit  following the manufacturer's instructions.The content of Gag.MS2 precursor proteins in Gag.MS2 supernatants was analyzed by an immunoblot assay. In brief, 15 μL of each supernatant were mixed with 15 μL of Laemmli buffer and denatured for 5 min at 95°C. Next, 20 μL of each sample was separated by an SDS-PAGE (12.5% separation gel) and transferred to a nitrocellulose membrane . Successful blotting and equal sample loading were controlled by staining the membranes with Ponceau S solution . Afterward, the membranes were probed with a polyclonal rabbit-anti-enterobacteria phage MS2 coat protein antibody  in Tris-buffered saline with 0.05% (v/v) Tween and 3% milk powder . As a secondary antibody, a goat-anti-rabbit immunoglobulin G (IgG)  conjugated with horseradish peroxidase was used. Final visualization and quantification were carried out by treating the membrane with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and the FUSION FX imaging system , respectively.In graph of


D1CreERT2GluA1 or D1CreERT2GluN1mice) have altered sugar consumption in both sexes, whereas DATCreERT2GluA1 and DATCreERT2GluN1do not differ from their respective littermate controls. In terms of relapse-like behavior, female D1CreERT2GluN1mice show a more pronounced SDE. Given that glutamate receptors within the mesolimbic system play a critical role in mediating relapse behavior of alcohol and other drugs of abuse, it is surprising that these receptors do not mediate the SDE, or in the case of female D1CreERT2GluN1 mice, show an opposing effect. We conclude that a relapse-like phenotype of sugar consumption differs from that of drugs of abuse on the molecular level, at least with respect to the contribution of mesolimbic glutamate receptors.Glutamatergic input via NMDA and AMPA receptors within the mesolimbic dopamine (DA) pathway plays a critical role in the development of addictive behavior and relapse toward drugs of abuse. Although well-established for drugs of abuse, it is not clear whether glutamate receptors within the mesolimbic system are involved in mediating chronic consumption and relapse following abstinence from a non-drug reward. Here, we evaluated the contribution of mesolimbic glutamate receptors in mediating chronic sugar consumption and the sugar-deprivation effect (SDE), which is used as a measure of relapse-like behavior following abstinence. We studied four inducible mutant mouse lines lacking the GluA1 or GluN1 subunit in either DA transporter (DAT) or D1R-expressing neurons in an automated monitoring system for free-choice sugar drinking in the home cage. Mice lacking either GluA1 or GluN1 in D1R-expressing neurons ( Here, we provide evidence from various inducible and site-specific transgenic mouse models that glutamate receptors within the mesolimbic dopamine (DA) system do not play a critical role in relapse behavior in chronically sugar-drinking male and female mice. This differentiates a natural reward from drugs of abuse on the molecular level, as mesolimbic NMDA and AMPA receptors are essential for drug-induced neuroplasticity and subsequent relapse behavior.It is assumed that the problematic chronic use of sugar, similar to chronic consumption of drugs of abuse, can lead to an addictive-like phenotype. However, the concept of “sugar addiction” is controversial and only a few studies have attempted to determine the addictive properties of sugar using rigorous scientific criteria .These studies suggest that behavioral phenotypes associated with chronic consumption of drugs of abuse and sugar consumption are similar with respect to withdrawal responses, compulsive over-consumption, craving, and loss of control . After dGiven the similarities of phenotypes for the chronic use of drugs of abuse and sugar, we speculated that there may also be similarities on the molecular level. In the addiction field, there is strong evidence that an interaction between the glutamatergic and mesolimbic dopamine (DA) systems is critical for mediating the reinforcing effects of drugs of abuse and consequently addictive behavior and relapse . In partSome drug-induced neuroplastic changes within the mesolimbic system may also occur following consumption of natural rewards. For example, sucrose intake increases the phosphorylation and trafficking of accumbal AMPA receptor GluA1 subunits  and alteSlc6a3) or D1 (Drd1a) promoter following the previously described procedure  or D1 (Drd1a) promoter following the previously described procedure  into a bacterial artificial chromosome containing the gene encoding DAT (Slc6a3) or D1 (Drd1a) by recombineering. fl/flGluN1 and fl/flGluA1 mice, having exons 11–18 of the Grin1 or exon 11 of the Gria1 alleles, respectively, flanked with loxP sites were generated by gene targeting in embryonic stem cells . As controls, floxed littermates not carrying the Cre-recombinase were used.We generated mutant mice expressing GluN1 or GluA1 mutations under control of the DAT  and Germany (Deutsches Tierschutzgesetz) and the experiment was approved by the German animal welfare authorities (Regierungspräsidium Karlsruhe).Mice were single-housed in standard hanging cages at 21 ± 1°C and 50 ± 5% relative humidity on a reversed 12/12 h light/dark cycle, with lights on at 7:30 P.M. The animals were provided with standard rodent food , a bottle containing 5% (w/v) sugar solution during the long-term sugar paradigms (see below for details) and tap water DATCreERT2GluN1, 41 D1CreERT2GluN1, 53 DATCreERT2GluA1, 37 D1CreERT2GluA1, and 180 respective control littermates  were used. Mice had continuous free-choice access to a bottle containing a sugar solution (sucrose 5% w/v) and a bottle with tap water in the homecage for eight weeks. During the last 3 d of sugar exposure, sugar and water intake and locomotion were recorded using a drinkometer system  and water (ml) intake, sugar preference  and locomotor activity were calculated per day. During baseline and SDE measurements, sugar and water intake and locomotion were additionally calculated in 4-h time intervals. Baselines were calculated as the mean of the last 3 d of baseline recording.post hoc comparisons using Statistica 10 (StatSoft). All values are given as mean ± SEM, and statistical significance was set at p < 0.05.Statistical analyses were performed by one-way or two-way ANOVA with repeated measures and Newman–Keuls test for The ANOVA model for the long-term free-choice home cage drinking and SDE contained the fixed effects of sugar deprivation (baseline and relapse), gene (wild-type and GluA1 or GluN1), and the interaction deprivation × gene.DATCreERT2GluN1mice did no differ from their wild-type littermates in the daily sugar intake  = 0.06, p = 0.8 and for F = 2.7, p = 0.1). Male DATCreERT2GluN1mice displayed a decreased baseline locomotion compared with wild-types  = 4.4, p < 0.05;F = 0.2, p = 0.7;After eight weeks of chronic, 24-h free-choice sugar exposure, male and female F = 62.3, p < 0.0001 and for F = 72.8, p < 0.0001). The dissection of the baseline and SDE drinking into 4-h time interval points showed the typical diurnal pattern of intake, characterized by higher drinking during the dark, active phase, and lower drinking during the light, inactive phase of the day. Specifically, during the SDE , sugar intake was strongly pronounced during the first 4–8 h of re-exposure and lasted not longer than 24 h in all genotypes  = 1.3, p = 0.7 and for F = 0.07, p = 0.8, Neuman–Keuls post hoc p < 0.001). A similar diurnal pattern was observed in the locomotor activity  = 58.5, p < 0.0001 and for F = 66.2, p < 0.0001), by increasing locomotion in mutant males and in wild-type females at the end and the beginning of the SDE, respectively, a phenomenon not related to GluN1 mutations  = 1.6, p = 0.2 and for F = 2.3, p = 0.1, Neuman–Keuls post hoc p < 0.001). Supporting these results, the percentage of relapse over baseline during the first 4 h of sugar re-exposure for intake and locomotion  = 1.7, p = 0.2 and for F = 3.8, p = 0.06; for F = 2.5, p = 0.1 and for F = 2.5, p = 0.1).A period of sugar deprivation significantly increased the sugar intake in all mutants, and the respective wild-type mice C,I, indiactivity D,J durincomotion E,K, F,L GluN1 mutation onto D1-containing neurons (D1CreERT2GluN1mice) had an effect on chronic sugar drinking. As depicted in D1CreERT2GluN1mice showed a significant decrease in the total, 24-h free-choice sugar drinking  = 4.5, p < 0.05 and for F = 5.9, p < 0.05). No differences were found between genotypes in locomotor activity at baseline  = 1.3, p = 0.3 and F = 0.001, p = 1).In contrast to the DAT-containing neurons, GluN1 mutation onto D1-containing neurons (D1CreERT2GluN1 mice) had an impact on the SDE. Thus, although a period of sugar deprivation significantly increased the sugar intake in all mice  = 63.5, p < 0.0001 and for F = 66.6, p < 0.0001), statistical analysis also showed a gene × deprivation interaction effect  = 4.4, p < 0.0001 and for F = 1.9, p < 0.05, Neuman–Keuls post hoc p < 0.05). Again, the SDE was strongly pronounced during the first 4–8 h of re-exposure and lasted not longer than 24 h in all mice. Locomotor activity  = 55.3, p < 0.0001, Neuman–Keuls post hoc p < 0.05 and for F = 54.2, p < 0.0001). When calculating the percentage of relapse over baseline during the first 4 h of sugar re-exposure for intake and locomotion, male D1CreERT2GluN1mice did not differ from their wild-type littermates  = 1.9, p = 0.2 and for F = 0.3, p = 0.6). However, in females, the SDE magnitude was higher for the intake  = 8, p < 0.01) and lower for the locomotion  = 6.2, p < 0.05).Furthermore (and in contrast to the DAT-containing neurons), all mice C,I, indiactivity D,J was aDATCreERT2GluA1 mice did not differ from their respective wild-type littermates after eight weeks of chronic, 24-h free-choice sugar exposure. As shown in F = 0.1, p = 0.7 and for F = 1.3, p = 0.3) as well as locomotor activity  = 1.5, p = 0.2 and F = 0.1, p = 0.7) remained unaltered in the mutants.Deletion of the AMPA receptor GluA1 subunit onto DAT-containing neurons did not have any effect on chronic sugar drinking, as the phenotypes displayed by both male A,B and fGluN1 mutations. That is, all mice showed the typical diurnal pattern of consumption, and a strongly pronounced sugar intake during the first 4–8 h of re-exposure which lasted not longer than 24 h. Statistical analysis showed a deprivation effect for male  = 60.7, p < 0.0001, Neuman–Keuls post hoc p < 0.01) and female  = 85.2, p < 0.0001, Neuman–Keuls post hoc p < 0.01), which was not GluA1 dependent  = 2.4, p = 0.1 and for F = 1, p = 0.3). Locomotor activity did not differ between baseline and SDE  = 0.1, p = 0.7 and for F = 0.1, p = 0.8), although statistical analysis showed a deprivation effect  = 42, p < 0.001 and F = 70, p < 0.001). In line with these results, the percentage of relapse over baseline during the first 4 h of sugar re-exposure for intake and locomotion, did not differ in male  = 3.9, p = 0.06 and F = 1.1, p = 0.3) or female  = 0.7, p = 0.4 and F = 0.7, p = 0.4) DATCreERT2GluA1mice from their wild-type littermates.Deletion of the AMPA receptor GluA1 subunit onto DAT-containing neurons did not have a role in relapse to sugar, as measured by the SDE C–F, I–L. and SDE D,J for mGluN1 mutant mice, GluA1 mutation onto D1-containing neurons (in contrast to the DAT-containing neurons), had an effect on chronic sugar drinking behavior  = 4.3, p < 0.05 and F = 17.1, p < 0.001). Regarding locomotor activity, it did not change in male mutants  = 0.9, p = 0.3), but it was significantly increased in female D1CreERT2GluA1mice  = 6.9, p < 0.05).As observed with the behavior . That isGluA1 mutation onto D1-containing neurons did not influence the SDE  = 60.7, p < 0.001 and F = 73, p < 0.001, Neuman–Keuls post hoc p < 0.05). However, locomotor activity was not only influenced by a sugar deprivation period  = 55, p < 0.001 and F = 55.6, p < 0.001, Neuman–Keuls post hoc p < 0.05), but also by the GluA1 receptor  = 4.2, p < 0.05 and F = 12.3, p < 0.01; gene × deprivation interaction effect: F = 1.8, p < 0.001 and F = 4.6, p < 0.001) as only the male and female mutants showed an increased locomotor activity at the end of the SDE period. Calculating the percentage of relapse during the first 4 h of sugar re-exposure for intake and locomotion similar SDE magnitudes were found in both males  = 0.006, p = 1 and F = 0.06, p = 0.08) and females  = 2, p = 0.2 and F = 0.005, p = 0.9) compared with their respective wild-type littermates.Similar to the DAT-containing mutant mice,  the SDE , and allD1CreERT2GluN1 mice show a more pronounced relapse-like behavior in the SDE model.Here, we report on three findings in different inducible mouse mutant lines lacking either GluN1 or GluA1 receptor subunits in DAT or D1-containing neurons in a chronic free-choice sugar consumption paradigm and the SDE model. First, long-term sugar intake is modulated by AMPA and NMDA receptors in D1-containing neurons in an opposing manner. The specific deletion of the GluA1 subunit, which yields non-functional AMPA receptors in primarily D1-containing MSNs, increases excessive sugar drinking in male and female mice, whereas mice with inducible GluN1 receptor subunit deletion in D1-expressing neurons show significantly reduced chronic sugar intake. Second, neither AMPA nor NMDA receptors in DA neurons influence the development and maintenance of sugar consumption. Third, female The four genetic mouse models used here have some advantages over other approaches for gene targeting, allowing a more precise demonstration of the functional role regarding the gene of interest. First, these mutant models have high specificity of the deletion of GluN1 or GluA1 in DAT-expressing or D1-expressing neurons, as shown by previous co-localization studies . Cre-expD1CreERT2GluA1 male and female mice, suggesting that functional AMPA receptors onto D1-containing neurons play a role in the regulation of excessive sugar consumption. Our finding largely agrees with a previous report that studied the regulation of AMPA receptors on NAc synapses by sucrose intake. DATCreERT2GluN1 and D1CreERT2GluN1 mutant mice showed no change in sugar binging suggest that non-mesolimbic brain regions may also contribute to sugar binging.As previously reported , mice shD1CreERT2GluN1 mutant mice showed a more pronounced SDE. In contrast, relapse behavior to drugs of abuse is strongly under the control of mesolimbic glutamate receptors, especially the ADE, like the SDE a measure of relapse-like behavior (D1CreERT2GluN1 female mutant mice exhibit an augmented SDE.In terms of relapse-like behavior, we tested the four mutant mouse lines in the SDE model and found that neither AMPA nor NMDA receptors in DA neurons influenced augmented sugar consumption following a deprivation period. However, female behavior . All foubehavior . Similarbehavior  and a vabehavior . For othbehavior  and delebehavior . In concIn summary, a previous study demonstrated the occurrence of an addictive-like phenotype for sugar in male and female mice similar to that of drugs of abuse . Here, w


Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies.Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean , with those exhibiting visible mutant phenotypes showing higher frequencies (27–100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants.We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used ‘half-seed’ explants derived from imbibed seeds for Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.The online version contains supplementary material available at 10.1186/s12896-022-00737-7. Streptococcus pyogenes is among the best characterized. It is consisted of nuclease Cas9, crRNA, and an auxiliary trans-activating crRNA (tracrRNA) required for processing of crRNA into functioning units [Streptococcus pyogenes [Genome editing using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system has emerged as a versatile tool to modify genes at precise locations. It was initially discovered as bacterial immune system against viruses and other foreign nucleic acids such as plasmids –5. CRISPng units , 10. In ng units , 11. Caspyogenes , 11–13. pyogenes , 9. The pyogenes , 13. Oncpyogenes , apple [pyogenes , tomato pyogenes , grape [pyogenes , 18, melpyogenes , etc. ThGlycine max L. Merr.) is a globally important crop that provides a rich source of protein and oil. Since year 2015, genome editing in soybean has been performed by several research groups, often using hairy root system [Agrobacterium-mediated transformation [Soybean  genes for editing through NHEJ, due to the distinctive phenotypes of the loss of function mutant. In Arabidopsis, PDS encodes an enzyme in the carotenoid biosynthesis pathway, disruption of this gene results dwarf and albino phenotypes , making Agrobacterium-mediated genetic transformation was performed using ‘half-seed’ explants based on the methodology described by Paz et al. [Soybean is a palaeopolyploid organism which underwent genome duplication in ancient times, nearly 75% of its genes are in multiple copies . The soyz et al.  to delivz et al. .Table 1OWe recovered two plantlets from GmPDS1 and eight plantlets from GmPDS3 transformation procedures. To verify transformation, genomic DNA was extracted from leaf tissues and used for polymerase chain reaction (PCR) using construct-specific primers , and the rest were all independent lines, denoted by different numbers. Mutations were detected in 9 out of 10 transgenic plants in both PDS genes, corresponding to 90% mutation efficiency  as well as two homologous GIGANTEA genes (Glyma.10g221500 and Glyma.20g170000). They are hereafter referred as ARF10, ARF13, GGT10, and GGT20, respectively. It is worth noting that all of these sites are followed by PAM ‘AGG’. ARF10 and ARF13 contain 5 and 10 consecutive mismatches to GmPDS8 target sequence, located immediately upstream of PAM. GGT10 and GGT20 have 7 and 6 interspaced mismatches, respectively; both contain 4 consecutive mismatches directly upstream of PAM , #5 7), #8 (2), #13 (5), and #15 (8) were planted in soil, with respective quantities for each line indicated in brackets. Due to developmental defects, seeds from line #3 and #8 did not germinate. A total of six T1 line #5 plants grew in soil, among which three  exhibited strong dwarf and albino phenotypes, while others  had phenotypes indistinguishable from wild type ,All T1 line #13 and #15 seeds germinated and developed into plants showing wild type phenotypes. The transgene was not detected in these plants. Targeted fragments from plant #13–1, 13–2, 13–3, 15–1, 15–2, 15–3, and 15–4 were sequenced, and no mutation was detected  (Difco) medium containing 50 mg/L kanamycin and 25 mg/L rifampicin (Sigma-Aldrich) were randomly selected for verification by PCR using vector specific primers  medium ethanesulfonic acid (MES), 3% sucrose, 7.5 μM 6-benzylaminopurine (BAP), 0.7 μM gibberellic acid (GA), 200 μM acetosyringone, pH 5.4) , and resuspended in half of the original volume in LCC medium, for a final O.D.600 of 1.5.Genome editing constructs were transferred into 2O, at room temperature in dark. On the day of transformation, seed coat was removed, and each embryo was cut longitudinally along the hilum, resulting separate cotyledons attached to the halved embryo axis. Small axillary shoots, if present, were removed, and the node at the junction of cotyledon and embryo axis was gently wounded with a scalpel. In each 100 × 20 mm petri dish, about 60 explants were immersed for 30 min in infection broth containing Agrobacterium carrying the constructs, at room temperature with gentle shaking at 50 rpm. Afterwards, explants were blotted dry on sterile filter paper, and placed flat side down on co-cultivation medium , 158 mg/L sodium-thiosulfate, 200 μM acetosyringone, pH 5.4 with 0.7% agar) for 5 days. Tissue culture was carried out at 24 °C under 16-h photoperiod with 100μmoles/s/m2 illumination. Following co-cultivation, explants were washed 2 times in wash medium , and transferred to shoot inducing medium (SIM)  initially without selection for two weeks, placed flat side up with the base section embedded in the medium. Subsequently, explants were transferred to SIM containing 6-10 mg/L herbicide glufosinate-ammonium, also known as BASTA, for selection. Surviving shoot buds along with the base were removed from the original explants, and cultured on shoot elongation medium (SEM) , 50 mg/L L-asparagine monohydrate, 100 mg/L L-pyroglutamic acid, 75 mg/L cefotaxime, 300 mg/L timentin, 5 mg/L BASTA, pH 5.6, with 0.7% agar). Elongated shoots (> 2 cm) were excised from shoot pads, dipped in sterile 0.5–1 mg/mL indole-3-butyric acid (IBA) and transferred to the rooting medium (RM) . Rooted plantlets were separated from agar, rinsed with water, and transplanted to PRO-MIX BX soil, grown in growth cabinets at 24 °C under 16-h photoperiod with 200μmoles/s/m2 illumination.Soybean cv. Williams 82 seeds were kindly provided by Dr. Aiming Wang at Agriculture and Agri-Food Canada. Seeds were surface sterilized overnight in a desiccator using chlorine gas produced by mixing 5 mL of 12 N hydrochloric acid (HCl) (Sigma-Aldrich) with commercial bleach containing 6% (w/v) sodium hypochlorite. Transformation procedures were adapted from Paz et al.  and Olho2O. To verify transformation, genomic DNA was used as template for PCR using Promega GoTaq Flexi taq DNA polymerase with construct-specific primers  according to manufacturer’s procedures. Extracted plasmids were sent to Eurofins Genomics for Sanger sequencing.To extract total genomic DNA, leaf tissues from plants growing in soil or from regenerating shoots were collected and homogenized using Qiagen Tissue Lyser II homogenizer. Homogenized tissue was resuspended in DNA extraction buffer made of 2% hexadecyltrimethylammonium bromide (CTAB), 100 mM Tris pH 8.0, 20 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1.4 M sodium chloride (NaCl), 2% polyvinylpyrrolidone (PVP)-40 (Sigma-Aldrich), followed by centrifugation at 5000 rpm for 10 min to settle the debris. The supernatant was incubated at 65 °C for 1 h before mixed with 1 volume of chloroform:isoamyl alcohol (24:1) (Sigma-Aldrich), then centrifuged at 13000 rpm for 10 min to separate the aqueous and organic phases. The aqueous phase was mixed with 1 volume of isopropanol (Sigma-Aldrich) for DNA precipitation. Following centrifugation at 13000 rpm for 10 min at 4 °C, DNA pellet was washed with 70% ethanol. The final DNA pellet was dried in a 60 °C oven for 10 min, and resuspended in sterile ddHE.coli clones, according to procedures described above.The 20-nt GmPDS8 guide sequence was used as query for nucleotide BLAST provided by National Center for Biotechnology Information (NCBI) , 56 to sNucleotide and peptide sequences of GmPDS11g and GmPDS18g coding regions were aligned using MegAlign Pro 17 software, using ClustalW method.Additional file 1.Figure S1. Alignment of GmPDS11g and GmPDS18g nucleotide coding sequences.Additional file 2.Figure S2. Alignment of GmPDS11g and GmPDS18g peptide sequences.Additional file 3.Table S1. Summary of soybean genetic transformations with genome editing constructs.Additional file 4.Table S2. Detection of mutations in T0 plants.Additional file 5.Table S3. Detection of mutations in GmPDS8 T1 plants.Additional file 6.Table S4. Off-target analysis in GmPDS8 T0 plants.Additional file 7.Table S5. Primers used for cloning and molecular analyses.Additional file 8.Figure S3. Example agarose gels showing detection of transgene in GmPDS8 T1 plants.