Exogenous γ-aminobutyric acid (GABA) alleviates nitrogen deficiency by mediating nitrate uptake and assimilation in Andrographis paniculata seedlings.

γ-Aminobutyric acid (GABA) plays significant metabolic and signaling roles in plant stress responses. Recent studies have proposed that GABA alleviates plant nitrogen (N) deficient stress; however, the mechanism by which GABA mediates plant N deficiency adaptation remains not yet well understood. Herein we found in a medicinal plant Andrographis paniculata that 5 mmol L-1 exogenous GABA promoted plant growth under N deficient (1 mmol L-1 NO3-) condition, with remarkably increments in total N and NO3- concentrations in plants. GABA increased N assimilation and protein synthesis by up-regulating the activities and expression of N metabolic enzymes. GABA also increased the accumulation of α-ketoglutarate and malate, which could facilitate the assimilation of NO3-. Inhibition of NR by Na2WO4 counteracted the promoting effects of GABA on plant growth, and the effects of GABA were not affected by L-DABA and 3-MP, the inhibitors of GABA transaminase (GABA-T) and glutamate decarboxylase (GAD), respectively. These results suggested that the nutritional role of GABA was excluded in promoting plant growth under low N condition. The results of 15N isotopic tracing and NRTs transcription indicated that exogenous GABA could up-regulate NRT2.4 and NRT3.2 to increase plant NO3- uptake under N deficient condition. Interestingly, primidone, an inhibitor of GABA receptor, impeded the effects of GABA on plant growth and N accumulation. Thus, our results revealed that exogenous GABA acted as a signal to up-regulate NRTs via its receptor to increase NO3- uptake, and subsequently promoted NO3- assimilation to alleviate N deficiency in A. paniculata.

Loss of function of CMPK2 causes mitochondria deficiency and brain calcification.

Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.

Concentration-dependent dual effects of exogenous sucrose on nitrogen metabolism in Andrographis paniculata.

The effects of exogenous sucrose (Suc) concentrations (0, 0.5, 1, 5, 10 mmol L-1) on carbon (C) and nitrogen (N) metabolisms were investigated in a medicinal plant Andrographis paniculata (Chuanxinlian). Suc application with the concentration of 0.5-5 mmol L-1 significantly promoted plant growth. In contrast, 10 mmol L-1 Suc retarded plant growth and increased contents of anthocyanin and MDA and activity of SOD in comparison to 0.5-5 mmol L-1 Suc. Suc application increased contents of leaf soluble sugar, reducing sugar and trerhalose, as well as isocitrate dehydrogenase (ICDH) activity, increasing supply of C-skeleton for N assimilation. However, total leaf N was peaked at 1 mmol L-1 Suc, which was consistent with root activity, suggesting that exogenous Suc enhanced root N uptake. At 10 mmol L-1 Suc, total leaf N and activities of glutamine synthase (GS), glutamate synthase (GOGAT), NADH-dependent glutamate dehydrogenase (NADH-GDH) and glutamic-pyruvic transaminase (GPT) were strongly reduced but NH4+ concentration was significantly increased. The results revealed that exogenous Suc is an effective stimulant for A. paniculata plant growth. Low Suc concentration (e.g. 1 mmol L-1) increased supply of C-skeleton and promoted N uptake and assimilation in A. paniculata plant, whereas high Suc concentration (e.g. 10 mmol L-1) uncoupled C and N metabolisms, reduced N metabolism and induced plant senescence.

Sulfur Regulates the Trade-Off Between Growth and Andrographolide Accumulation via Nitrogen Metabolism in Andrographis paniculata.

Nitrogen (N) and sulfur (S) are essential mineral nutrients for plant growth and metabolism. Here, we investigated their interaction in plant growth and andrographolide accumulation in medicinal plant Andrographis paniculata grown at different N (4 and 8 mmol·L-1) and S concentration levels (0.1 and 2.4 mmol L-1). We found that increasing the S application rate enhanced the accumulation of andrographolide compounds (AGCs) in A. paniculata. Simultaneously, salicylic acid (SA) and gibberellic acid 4 (GA4) concentrations were increased but trehalose/trehalose 6-phosphate (Tre/Tre6P) concentrations were decreased by high S, suggesting that they were involved in the S-mediated accumulation of AGCs. However, S affected plant growth differentially at different N levels. Metabolite analysis revealed that high S induced increases in the tricarboxylic acid (TCA) cycle and photorespiration under low N conditions, which promoted N assimilation and S metabolism, and simultaneously increased carbohydrate consumption and inhibited plant growth. In contrast, high S reduced N and S concentrations in plants and promoted plant growth under high N conditions. Taken together, the results indicated that increasing the S application rate is an effective strategy to improve AGC accumulation in A. paniculata. Nevertheless, the interaction of N and S affected the trade-off between plant growth and AGC accumulation, in which N metabolism plays a key role.

Organic nitrogen sources promote andrographolide biosynthesis by reducing nitrogen metabolism and increasing carbon accumulation in Andrographis paniculata.

Nitrogen (N) form affects secondary metabolites of medicinal plants, but the physiological and molecular mechanisms remain largely unknown. To fully understand the response of andrographolide biosynthesis to different N forms in Andrographis paniculata, the plants were fed with nutritional solution containing sole N source of nitrate (NO3-), ammonium (NH4+), urea or glycine (Gly), and the growth, carbon (C) and N metabolisms and andrographolide biosynthesis were analyzed. We found that plants grown in urea and Gly performed greater photosynthetic rate and photosynthetic N use efficiency (PNUE) than those grown in NO3- and NH4+. Organic N sources reduced the activities of enzymes involving in C and N metabolisms such as glutamine synthase (GS), glutamate synthase (GOGAT) and NADH-dependent glutamate dehydrogenase (NADH-GDH), invertase (INV), isocitrate dehydrogenase (ICDH) and glycolate oxidase (GO), resulting in reduced depletion of carbohydrates and increased starch accumulation. However, they enhanced andrographolide content by up-regulating the key genes in its biosynthetic pathway including HMGR, DXS, GGPS and ApCPS. Besides, NH4+ decreased leaf SPAD value, contents of soluble protein and amino acids and GO activity, but increased photosynthetic rate and contents of soluble sugar and starch in comparison to NO3-. Andrographolide biosynthesis was also up-regulated. The results revealed that increasing accumulation of carbohydrates, especially starch, was beneficial to the biosynthesis of andrographolide; organic N sources decreased carbohydrate depletion by reducing N metabolism, and promoted plant growth and andrographolide biosynthesis synergistically.

Electron Microscopy-Based Comparison and Investigation of the Morphology of Exosomes Derived from Hepatocellular Carcinoma Cells Isolated at Different Centrifugal Speeds.

Exosomes derived from hepatocellular carcinoma (HCC) cells are nanovesicles and are involved in the occurrence and development of HCC, they also serve as important carriers and drug targets of nanodrug delivery systems. The external shape and internal structure of exosomes are important indexes of identification, and isolated intact morphology is crucial to biological function integrity. However, given their susceptibility to various influencing factors, the external shape and internal structure of exosomes derived from HCC cells remain incompletely studied. In this study, exosomes purified from HCC cells were isolated at different centrifugation speeds and examined via multiple electron microscopy (EM) techniques. The results demonstrate that exosomes possess a nearly spherical shape and bilipid membranous vesicle with a concave cavity structure containing electron-dense and coated vesicles, suggesting the possible existence of subpopulations of exosomes with specific functions. The exosomes isolated at ultracentrifugation (UC) speed (≥110,000×g) presented irregular and diverse external morphologies, indicating the effect on the integrity of the exosomes. Transforming growth factor signaling bioactive substances (TGF-ß1, S100A8, and S100A9) can be found in exosomes by performing Western blotting, showing that the internal content is associated with metastasis of HCC. These findings show that EMelectron microscopy and UC speed can affect exosome characteristics, including external shape, internal structure, and content of bioactive substances. The electron-dense and coated vesicles that had been discovered in exosomes might become new additional morphological features, which could help to improve the interpretation of experimental results and widen our understanding of exosome morphology.

Distribution, diastereomer-specific accumulation and associated health risks of hexabromocyclododecanes (HBCDs) in soil-vegetable system of the Pearl River Delta region, South China.

The distribution and diastereomeric profiles of hexabromocyclododecanes (HBCDs, identified as persistent organic pollutants) in soil-vegetable system of open fields remain unknown. In this study, three main HBCD diastereoisomers (α-, ß-, and γ-HBCDs) were analyzed in paired soil and vegetable samples from vegetable farms in four cities (Guangzhou, Jiangmen, Huizhou, Foshan) of the Pearl River Delta region, Southern China. The sum concentrations of the three diastereoisomers (∑HBCDs) in soils varied from 0.99 to 18.4 ng/g (dry weight) with a mean of 5.77 ng/g, decreasing in the order of Jiangmen > Guangzhou > Huizhou > Foshan. The distributions of HBCDs in both soil and vegetable were diastereomer-specific, with γ-HBCD being predominant. The ∑HBCDs in vegetables ranged from 0.87 to 32.7 ng/g (dry weight) with a mean of 16.6 ng/g, generally higher than those of the corresponding soils. Thus bioconcentration factors (BCFs, the ratio of contaminant concentration in vegetable to that in soil) of HBCDs were generally greater than 1.0, implying higher accumulation in vegetable. The estimated daily intake (EDI) of ΣHBCDs via consumption of vegetables varied from 0.26 to 9.35 ng/kg bw/day with a mean of 3.60 ng/kg bw/day for adults and from 0.32 to 11.5 ng/kg bw/day with a mean of 4.41 ng/kg bw/day for Children, far lower than the oral reference dose (RfD, 2 × 105 ng/kg bw/day) proposed by US National Research Council. These results suggest that HBCD in the vegetables posed low health risk for the local population. These data are the first report on HBCD occurrence and health risk in soil-vegetable system of open fields.

Bioaugmentation of Exogenous Strain Rhodococcus sp. 2G Can Efficiently Mitigate Di(2-ethylhexyl) Phthalate Contamination to Vegetable Cultivation.

This work developed a bioaugmentation strategy that simultaneously reduced soil di(2-ethylhexyl) phthalate (DEHP) pollution and its bioaccumulation in Brassica parachinensis by inoculating the isolated strain Rhodococcus sp. 2G. This strain could efficiently degrade DEHP at a wide concentration range from 50 to 1600 mg/L and transformed DEHP through a unique biochemical degradation pathway that distinguished it from other Rhodococcus species. Besides, strain 2G colonized well in the rhizosphere soil of the inoculated vegetable without competition with indigenous microbes, resulting in increased removal of DEHP from soil (∼95%) and reduced DEHP bioaccumulation in vegetables (∼75% in the edible part) synchronously. Improved enzyme activities and DOC content in the rhizosphere of the planting vegetable and inoculating strain 2G were responsible for the high efficiency in mitigating DEHP contamination to vegetable cultivation. This work demonstrated a great potential application to grow vegetables in contaminated soil for safe food production.

Biodegradation of di-butyl phthalate (DBP) by a novel endophytic bacterium Bacillus subtilis and its bioaugmentation for removing DBP from vegetation slurry.

Di-butyl phthalate (DBP) is a widely used plasticizer, recalcitrant and hazardous organic compound with high detection frequencies and concentrations in water and soil that pose a great threat to human health. A novel endphytic bacterium strain N-1 capable of efficiently degrading DBP and utilizing it as sole carbon source was isolated from Ageratum conyzoides. This bacterium was identified as Bacillus subtilis based on its morphological characteristics and 16S rDNA sequence analysis. Under the optimal culture conditions (pH 7.0, 30 °C), degradation percentage of DBP (12.5-100 mg/L) was up to 95% within five days, and its biodegradation half-life was less than 7.23 h. Degradation percentage of high DBP concentration (200 mg/L) was relatively lower (89%) with half-life of 56.8 h. DBP was degraded by Bacillus subtilis N-1 into mono-butyl phthalate and phthalic acid as evidenced by GC-MS analysis. Bioaugmentation of Youngia japonica plant slurry with strain N-1 greatly accelerated DBP dissipation with 97.5% removal percentage (higher by 47% than non-inoculation). The results highlighted that strain N-1 has great potential for bioremediation by plant-endophyte partnerships and for lowering PAE accumulation in crops.

Relationship between CT grouping and complications of liver cirrhosis.

BACKGROUND: Imaging examination is important for hepatic cirrhosis. But the relationship between magnetic resonance (MR), computed tomography (CT) or ultrasound findings and pathological groups, degree, or reserve function of the cirrhotic liver is not clear. In this study, we investigated the relationship between the CT groupings of liver cirrhosis and its complications and clinical conditions. METHODS: The CT findings in 357 patients with liver cirrhosis were grouped. The complications were analyzed, included splenomegaly, varicose collateral veins, ascites, pleurorrhea, primary liver carcinoma, gallbladder stone, etc. Blood routine (BRt), and serum usea nitrogen (SUN), creatinine and uric acid were measured and hypersplenia and liver-kidney syndrome were estimated. RESULTS: Three hundred and fifty-seven patients with cirrhosis were divided into homogeneous group (87 patients, 24.4%), segmental group (41, 11.5%), and nodal group (229, 64.2%). The grade of spleen enlargement in the segmental and the nodal groups was significantly greater than that in the homogeneous group (P<0.01 and P<0.001). The patients with varices were shown in a descending order in the segmental group (70.7%), the nodal group (17.0%) and the homogeneous group (2.3%), respectively. Significant difference was observed among the 3 groups (P<0.001). Ascites was seen in 182 patients (79.5%) of the nodal group, in 11 patients (26.8%) of the segmental group and in 9 patients (10.3%) of the homogeneous group (P<0.001). Sixty-eight patients (29.7%) in the nodal group had primary liver carcinoma and 1 (2.4%) in the segmental group and 5 (5.8%) in the homogeneous group (P<0.001). The number of patients with decreased concentration of hematoglobin in the nodal group was more than that in the homogeneous group (P<0.001). The mean values of hematoglobin and platelet in the nodal group and the segmental group were significantly lower than those in the homogeneous group (P<0.05). The number of patients with increased concentration of SUN in the nodal group and the segmental group was more than that in the homogeneous group (P<0.005). The concentration of SUN in the nodal group was significantly higher than that in the homogeneous group (P<0.002). CONCLUSION: There is a close relationship between the grouping of liver cirrhosis by CT findings and complications caused by the cirrhosis. The grouping is significant for estimating clinical conditions.

[A study on relationship between CT grouping and complication of liver cirrhosis].

OBJECTIVES: To probe into the relationship between the CT grouping of liver cirrhosis and its complications as well as clinical condition. METHODS: On the basis of CT findings in 357 cases with liver cirrhosis, CT grouping of the cirrhosis was performed. The complications of the cirrhosis, including enlargement of spleen, varicose collateral veins, ascites, pleurorrhea, primary liver carcinoma, gallbladder stone, etc, of all groups were analyzed. According to examination the blood routine, and the serum urea nitrogen (SUN), creatinine and uric acid, the condition of spleen function accentuation and liver-kidney syndrome were estimated. RESULTS: Three hundred and fifty-seven cases with cirrhosis were divided into homogeneous group (87 cases, 24.4%), segmental group (41 cases, 11.5%) and nodal group (229 cases, 64.1%). The grade of spleen enlargement in the segmental group and the nodal group was significantly greater than that in homogeneous group. The cases with varicose in the segmental group was the most (70.7%), in the nodal group next (17.0%) and in the homogeneous group the least (2.3%). There was significant difference among three groups. In the nodal group, there was ascites in 182 cases (79.5%) and significantly more than that in the segmental group (11 cases, 26.8%) and the homogeneous group (9 cases, 10.3%), and the former significantly more than the latter. There were 68 cases (29.7%) with primary liver carcinoma in the nodal group, and significantly more than that in the segmental group (1 case, 2.4%) and the homogeneous group (5 cases, 5.7%). The cases with hemoglobin decrease in the nodal group were significantly more than that in the homogeneous group. The averages of hemoglobin and platelet in the nodal group and the segmental group were significantly lower than that in the homogeneous group. The cases with SUN increase in the nodal group and the segmental group were significantly more than that in the homogeneous group. The concentration of SUN in the nodal group was significantly higher than that in the homogeneous group. CONCLUSIONS: There are close relationship between the grouping of liver cirrhosis on basis of CT findings and complications of the cirrhosis. The practice of grouping might be useful for estimating clinical condition