Efficacy and safety of compound glycyrrhizin in combination with conventional therapy in treatment of vitiligo: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Vitiligo is an acquired chronic depigmentary disorder affecting approximately 0.5% to 1% of individuals worldwide. The compound glycyrrhizin (CG), a complementary medicine, has been reported for treatment of vitiligo, but the evidence has not been systematically evaluated. We systematically assessed the efficacy and safety of CG in combination with conventional therapy for the treatment of vitiligo. METHODS: We searched Embase, Web of Science, PubMed, The Cochrane Library, Chinese BioMedical Literature Database (CBM), Wanfang Data, China National Knowledge Infrastructure (CNKI), and VIP information from inception to July 2022. Randomized controlled trials comparing CG combined with conventional therapy with conventional therapy alone for vitiligo were included in our analysis. The primary outcome was treatment response, which defined as >50% repigmentation rate of vitiligo after treatment. The secondary outcome was incidence of adverse events. Meta-analysis was performed using the Review Manager 5.4 software. Statistical heterogeneity was evaluated with chi-square and I2 statistics, dichotomous data were expressed as risk ratios (RR) with 95% confidence intervals using the Mantel-Haenszal method. RESULTS: Thirty-nine studies enrolling with 3994 participants were subjected to this review. The results of our meta-analysis indicated that addition of CG had superior effectiveness on repigmentation rate than phototherapy (RR = 1.28; P < .001), immunosuppressant (RR = 1.76; P < .001), traditional Chinese medicine (RR = 1.38; P < .001), combination of phototherapy and immunosuppressant (RR = 1.42; P < .001), and combination of phototherapy and traditional Chinese medicine (RR = 1.37; P < .001). In addition, CG did not increase the incidence of adverse events for vitiligo (RR = 0.79; P = .05). CONCLUSIONS: CG as a complementary medicine has a potential benefit in treatment of vitiligo. However, since the methodological flaws in the studies we included, more high-quality randomized controlled trials are warranted.

Calcium-dependent protein kinases CPK21 and CPK23 phosphorylate and activate the iron-regulated transporter IRT1 to regulate iron deficiency in Arabidopsis.

Iron (Fe) is an essential micronutrient for all organisms. Fe availability in the soil is usually much lower than that required for plant growth, and Fe deficiencies seriously restrict crop growth and yield. Calcium (Ca2+) is a second messenger in all eukaryotes; however, it remains largely unknown how Ca2+ regulates Fe deficiency. In this study, mutations in CPK21 and CPK23, which are two highly homologous calcium-dependent protein kinases, conferredimpaired growth and rootdevelopment under Fe-deficient conditions, whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions. Furthermore, we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRON-REGULATED TRANSPORTER1 (IRT1) at the Ser149 residue. Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity. Taken together, these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.

CBL1/9-CIPK23-NRAMP1 axis regulates manganese toxicity.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high-Mn-sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.

Isolation of Intact Vacuoles from Arabidopsis Root Protoplasts and Elemental Analysis.

The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins. Graphical abstract.

Understanding of protomers/deprotomers by combining mass spectrometry and computation.

Multifunctional compounds may form different prototropic isomers under different conditions, which are known as protomers/deprotomers. In biological systems, these protomer/deprotomer isomers affect the interaction modes and conformational landscape between compounds and enzymes and thus present different biological activities. Study on protomers/deprotomers is essentially the study on the acidity/basicity of each intramolecular functional group and its effect on molecular structure. In recent years, the combination of mass spectrometry (MS) and computational chemistry has been proven to be a powerful and effective means to study prototropic isomers. MS-based technologies are developed to discriminate and characterize protomers/deprotomers to provide structural information and monitor transformations, showing great superiority than other experimental methods. Computational chemistry is used to predict the thermodynamic stability of protomers/deprotomers, provide the simulated MS/MS spectra, infrared spectra, and calculate collision cross-section values. By comparing the theoretical data with the corresponding experimental results, the researchers can not only determine the protomer/deprotomer structure, but also investigate the structure-activity relationship in a given system. This review covers various MS methods and theoretical calculations and their devotion to isomer discrimination, structure identification, conformational transformation, and phase transition investigation of protomers/deprotomers.

Ca2+-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis in Arabidopsis.

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21/23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Ultra-fast screening of free fatty acids in human plasma using ion mobility mass spectrometry.

Free fatty acids are involved in many metabolic regulations in the human body. In this work, an ultra-fast screening method was developed for the analysis of free fatty acids using trapped ion mobility spectrometry coupled with mass spectrometry. Thirty-three free fatty acids possessing different unsaturation degrees and different carbon chain lengths were baseline separated and characterized within milliseconds. Saturated, monounsaturated, and polyunsaturated free fatty acids showed different linearities between collision cross-section values and m/z. The establishment of correlations between structures and collision cross-section values provided additional qualitative information and made it possible to determine free fatty acids which were out of the standards pool but possessed the confirmed linearity. The gas-phase separation made the quantitative analysis reliable and repeatable at a much lower time cost than chromatographic methods. The sensitivity was comparable to and even better than the reported results. The method was validated and applied to profiling free fatty acids in human plasma. Saturated free fatty acids abundance in the fasting state was found to be lower than that in the postprandial state, while unsaturated species abundance was found higher. The method was fast and robust with minimum sample pretreatment, so it was promising in the high-throughput screening of free fatty acids.

Ca2+-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis.

Manganese (Mn) is an essential micronutrient for all living organisms. However, excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development. Although a variety of Mn transporter families have been characterized, we have only a rudimentary understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants. Here, we demonstrate that two calcineurin-B-like proteins, CBL2/3, and their interacting kinases, CIPK3/9/26, are key regulators of plant Mn homeostasis. Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkably high Mn tolerance. Intriguingly, CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron (Fe) transporter MTP8 primarily at Ser35, which is conserved among MTP8 proteins from various species. Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole. Moreover, we show that sequential phosphorylation of MTP8, initially at Ser31/32 by the calcium-dependent protein kinase CPK5 and subsequently at Ser35 by CIPK26, provides an activation/deactivation fine-tuning mechanism for differential regulation of Mn transport. Collectively, our findings define a two-tiered calcium-controlled mechanism for dynamic regulation of Mn homeostasis under conditions of fluctuating Mn supply.

Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity. Despite their essential roles in Mn homeostasis, transcriptional and post-transcriptional modifications of Mn transporters remain poorly understood. Here, we demonstrated that high-Mn stress induces an obvious Ca2+ signature in Arabidopsis. We identified four calcium-dependent protein kinases, CPK4/5/6/11, that interact with the tonoplast-localized Mn and iron (Fe) transporter MTP8 in vitro and in vivo. The cpk4/5/6/11 quadruple mutant displayed a dramatic high-Mn-sensitive phenotype similar to that of the mtp8 mutant. CPKs phosphorylated the N-terminal domain of MTP8 primarily at the Ser31 and Ser32 residues. Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8S31/32A and phospho-mimetic variant MTP8S31/32D plants under different Mn and Fe conditions suggested that Ser31 and Ser32 are crucial for MTP8 function. In addition, genetic analysis showed that CPKs functioned upstream of MTP8. In summary, we identified a tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis and links Mn toxicity, Ca2+ signaling, and Mn transporters. These findings provide new insight into Mn homeostasis mechanisms and Ca2+ signaling pathways in plants, providing potential targets for engineering heavy metal toxicity-tolerant plants.

A novel role of the calcium sensor CBL1 in response to phosphate deficiency in Arabidopsis thaliana.

Phosphorus acts as an essential macroelement in plant growth and development. A lack of phosphate (Pi) in arable soil and phosphate fertilizer resources is a vital limiting factor in crop yields. Calcineurin B-like proteins (CBLs) act as one of the most important calcium sensors in plants; however, whether CBLs are involved in Pi deficiency signaling pathway remains largely elusive. In this study, we utilized a reverse genetic strategy to screen Arabidopsis thaliana T-DNA insertion mutants belonging to the CBL family under Pi deficiency conditions. The cbl1 mutant exhibited a relatively tolerant phenotype, with longer roots, lower anthocyanin content, and elevated Pi content under Pi deficiency, and a more sensitive phenotype to arsenate treatment compared with wild-type plants. Moreover, CBL1 was upregulated, and the mutation of CBL1 caused phosphate starvation-induced (PSIs) genes to be significantly induced under Pi deficiency. Histochemical staining demonstrated that the cbl1 mutant has decreased acid phosphatase activity and hydrogen peroxide concentrations under Pi deficiency. Collectively, our results have revealed a novel role of CBL1 in maintaining Pi homeostasis.

Analytical strategies for chemical characterization of bio-oil.

Bio-oils, produced by biomass pyrolysis, have become promising candidates for feedstocks of high value-added chemicals and alternative sources for transportation fuels. Bio-oil is such a complicated mixture that contains nonpolar hydrocarbons and polar components which cover almost all kinds of organic oxygenated compounds such as carboxylic acids, alcohols, aldehydes, ketones, esters, furfurals, phenolic compounds, sugar-like material, and lignin-derived compounds. Comprehensive characterization of bio-oil and its subfractions could provide insight into the conversion process of biomass processing, as well as its further utilization as transportation fuels or chemical raw materials. This review focuses on advanced analytical strategies on in-depth characterization of bio-oil, which is concerned with gas chromatography, high-resolution mass spectrometry, FTIR spectroscopy and NMR spectroscopy, offering complementary information for previous reviews.