The effect of intercropping leguminous green manure on theanine accumulation in the tea plant: A metagenomic analysis.

Intercropping is a widely recognised technique that contributes to agricultural sustainability. While intercropping leguminous green manure offers advantages for soil health and tea plants growth, the impact on the accumulation of theanine and soil nitrogen cycle are largely unknown. The levels of theanine, epigallocatechin gallate and soluble sugar in tea leaves increased by 52.87% and 40.98%, 22.80% and 6.17%, 22.22% and 29.04% in intercropping with soybean-Chinese milk vetch rotation and soybean alone, respectively. Additionally, intercropping significantly increased soil amino acidnitrogen content, enhanced extracellular enzyme activities, particularly ß-glucosidase and N-acetyl-glucosaminidase, as well as soil multifunctionality. Metagenomics analysis revealed that intercropping positively influenced the relative abundances of several potentially beneficial microorganisms, including Burkholderia, Mycolicibacterium and Paraburkholderia. Intercropping resulted in lower expression levels of nitrification genes, reducing soil mineral nitrogen loss and N2 O emissions. The expression of nrfA/H significantly increased in intercropping with soybean-Chinese milk vetch rotation. Structural equation model analysis demonstrated that the accumulation of theanine in tea leaves was directly influenced by the number of intercropping leguminous green manure species, soil ammonium nitrogen and amino acid nitrogen. In summary, the intercropping strategy, particularly intercropping with soybean-Chinese milk vetch rotation, could be a novel way for theanine accumulation.

Functional analysis of variants in DMD exon/intron 10 predicted to affect splicing.

Duchenne muscular dystrophy (DMD, MIM #310200) and Becker muscular dystrophy (BMD, MIM #300376) are X-linked recessive hereditary diseases caused by pathogenic variants in the DMD gene. Genetic testing of DMD identifies a certain number of variants of uncertain clinical significance (VUS) whose functional interpretations pose a challenge for gene-based diagnosis. To improve the accuracy of variant interpretation in public mutation repositories, we used computational tools to prioritize VUS and developed a cell-based minigene assay to confirm aberrant splicing. Using this procedure, we evaluated rare variants in exon and intron 10 of the DMD gene. We demonstrated that 16 variants, including both canonical and non-canonical splice sites, altered RNA splicing in variable patterns. Using the example of exon and intron 10 of the DMD gene, we demonstrated the utility of the in vitro minigene assay in the effective assessment of the spliceogenic effect for VUS identified in clinical practice and underlined the necessity of precise variant classification. This is the first systematic characterization of DMD splicing variants, besides, through our study, some undetermined variants are demonstrated to be pathogenic by altering RNA splicing of DMD.

Distribution of trace metals in a soil-tea leaves-tea infusion system: characteristics, translocation and health risk assessment.

The effects of metal pollution on tea are of great concern to consumers. We apply Geographic information systems technology to study the distribution of heavy metal elements in tea plantation ecosystems in Jiangsu Province, explore the relationships among metals in the soil, tea leaves and tea infusions, and assess the human safety risks of metals. The concentrations of nine metals in a soil-tea leaves-tea infusion system were studied at 100 randomly selected tea plantations in Jiangsu Province, China. Concentrations of selected metals, zinc (Zn), nickel (Ni), manganese (Mn), chromium (Cr) and copper (Cu), were quantified using an inductively coupled plasma-optical emission spectrometer (ICP-OES), and cadmium (Cd), arsenic (As), plumbum (Pb) and mercury (Hg) were quantified using inductively coupled plasma-mass spectrometry (ICP-MS). Arc-Map 10.3 was used for the spatial analysis of metals in soil, tea leaves and tea infusions. We found that the contents of Mn, Ni and Zn are high level in soil, tea leaves and tea infusions. The Mn level showed a spatial distribution pattern with greater concentrations at the junction of Nanjing and Yangzhou, southwest of Changzhou and west of Suzhou. The hazard index (HI) values in north-central Nanjing, southern Suzhou, southwestern Changzhou and northern Lianyungang were relatively greater. The Zn, Ni, Mn, Cr and Cu levels in the soil-tea infusion system were 17.3, 45.5, 54.5, 1.5 and 14.3%, respectively. The order of the leaching rates of the elements was Ni > Cr > Zn > Mn > Cu. The relative contribution ratios of HI were in the order of Mn > Ni > Cu > Zn > Cr > Pb > Cd > As > Hg. In tea infusions, the Mn level has the greatest potential health risks to consumers. Moreover, using Csoil it was inferred that the safety thresholds of Zn, Ni, Mn, Cr and Cu in soil were 27,700, 50, 1230, 493,000 and 16,800 mg L-1, respectively. The content of heavy metals in soil and tea varies greatly in different regions of Jiangsu Province, 92% of the soil has heavy metal content that meets the requirements of pollution-free tea gardens, 91% of tea samples met the requirements of green food tea. The thresholds for Ni (50 mg L-1) and Mn (1230 mg L-1) can be used as maximum limits in tea plantation soils. The consumption of tea infusions did not pose metal-related risks to human health.

The effects of tea plants-soybean intercropping on the secondary metabolites of tea plants by metabolomics analysis.

BACKGROUND: Intercropping, especially with legumes, as a productive and sustainable system, can promote plants growth and improves the soil quality than the sole crop, is an essential cultivation pattern in modern agricultural systems. However, the metabolic changes of secondary metabolites and the growth in tea plants during the processing of intercropping with soybean have not been fully analyzed. RESULTS: The secondary metabolomic of the tea plants were significant influence with intercropping soybean during the different growth stages. Especially in the profuse flowering stage of intercropping soybean, the biosynthesis of amino acids was significantly impacted, and the flavonoid biosynthesis, the flavone and flavonol biosynthesis also were changed. And the expression of metabolites associated with amino acids metabolism, particularly glutamate, glutamine, lysine and arginine were up-regulated, while the expression of the sucrose and D-Glucose-6P were down-regulated. Furthermore, the chlorophyll photosynthetic parameters and the photosynthetic activity of tea plants were higher in the tea plants-soybean intercropping system. CONCLUSIONS: These results strengthen our understanding of the metabolic mechanisms in tea plant's secondary metabolites under the tea plants-soybean intercropping system and demonstrate that the intercropping system of leguminous crops is greatly potential to improve tea quality. These may provide the basis for reducing the application of nitrogen fertilizer and improve the ecosystem in tea plantations.

Genome-wide identification, characterization and expression analysis of the amino acid permease gene family in tea plants (Camellia sinensis).

Amino acid permeases (AAPs) are involved in transporting a broad spectrum of amino acids and regulating physiological processes in plants. In this study, 19 AAP genes were identified from the tea plants genome database and named CsAAP1-19. Based on phylogenetic analysis, the CsAAP genes were classified into three groups, having significantly different structures and conserved motifs. In addition, an expression analysis revealed that most of CsAAP genes were specifically expressed in different tissues, especially CsAAP19 was expressed only in root. These genes also were significantly expressed in the Baiye 1 and Huangjinya cultivars. Nitrogen treatments indicated that the CsAAPs were obviously expressed in root. CsAAP2, -6, -12, -13 and - 16 were significantly expressed at 6 d after the glutamate treatment, while the expression trend at 24 h after contained the ammonium. These results improve our understanding of the CsAAP genes and their functions in nitrogen utilization in tea plants.

Genome-wide analysis of the SBP-box gene family transcription factors and their responses to abiotic stresses in tea (Camellia sinensis).

SQUAMOSA promoter-binding protein (SBP)-box gene family is one kind of plant-specific transcription factor that plays important roles in the process of resisting abiotic stress. The SBP-box gene family has been studied in many species, but their functions are not yet clear in Camellia sinensis var. sinensis (CSS) (tea) plants. In our study, 25 SBP-box genes in the CSS were identified in the reference genome and classified into six groups based on a phylogenetic tree. The expression pattern of CsSBP genes under temperature stresses showed that CsSBPs were involved in the process of resisting temperature stresses. CsSBP8 had a positive effect on the anthocyanin accumulation during high temperature exposures, but CsSBP12 has a high correlation with anthocyanin accumulation during both high and low temperature. This study provides a foundation for the further study of CsSBP genes involved in the anthocyanin biosynthesis pathway during the temperature stress in tea.

Genome-wide identification and expression analysis of the CLC superfamily genes in tea plants (Camellia sinensis).

The voltage-gated chloride channel (CLC) superfamily is one of the most important anion channels that is widely distributed in bacteria and plants. CLC is involved in transporting various anions such as chloride (Cl-) and fluoride (F-) in and out of cells. Although Camellia sinensis is a hyper-accumulated F plant, there is no studies on the CLC gene superfamily in the tea plant. Here, 8 CLC genes were identified from C. sinensis and they were named CsCLC1-8. The structure of CsCLC genes and the proteins were not conserved; the number of exons varied from 3 to 24, and the number of transmembrane domains contained 2 to 10. Furthermore, phylogenetic analysis revealed that CsCLC4-8 in subclass I contained the typical conserved domains GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), and CsCLC1-3 in subclass II did not contain any of the three conserved residues. We measured the expression levels of CsCLCs in roots, stems and leaves to assess the responses to different concentrations of Cl- and F-. The result indicated that CsCLCs participated in subfunctionalization in response to Cl- and F-, and CsCLC1-3 was more sensitive to F- treatments than CsCLC4-8, CsCLC6 and CsCLC7 may participate in absorption and long-distance transport of Cl-.

Genome-Wide Analysis Reveals Stress and Hormone Responsive Patterns of JAZ Family Genes in Camellia Sinensis.

JAZ (Jasmonate ZIM-domain) proteins play pervasive roles in plant development and defense reaction. However, limited information is known about the JAZ family in Camellia sinensis. In this study, 12 non-redundant JAZ genes were identified from the tea plant genome database. Phylogenetic analysis showed that the 12 JAZ proteins belong to three groups. The cis-elements in promoters of CsJAZ genes and CsJAZ proteins interaction networks were also analyzed. Quantitative RT-PCR analysis showed that 7 CsJAZ genes were preferentially expressed in roots. Furthermore, the CsJAZ expressions were differentially induced by cold, heat, polyethylene glycol (PEG), methyl jasmonate (MeJA), and gibberellin (GA) stimuli. The Pearson correlations analysis based on expression levels showed that the CsJAZ gene pairs were differentially expressed under different stresses, indicating that CsJAZs might exhibit synergistic effects in response to various stresses. Subcellular localization assay demonstrated that CsJAZ3, CsJAZ10, and CsJAZ11 fused proteins were localized in the cell nucleus. Additionally, the overexpression of CsJAZ3, CsJAZ10, and CsJAZ11 in E. coli enhanced the growth of recombinant cells under abiotic stresses. In summary, this study will facilitate the understanding of the CsJAZ family in Camellia sinensis and provide new insights into the molecular mechanism of tea plant response to abiotic stresses and hormonal stimuli.

Aluminum relieves fluoride stress through stimulation of organic acid production in Camellia sinensis.

Tea plants (Camellia sinensis O. Kuntze) can hyperaccumulate fluoride (F) in leaves. Although, aluminum (Al) can alleviate F toxicity in C. sinensis, the mechanisms driving this process remain unclear. Here, we measured root length, root activity, soluble proteins content, and levels of peroxidase, superoxide dismutase, catalase, malondialdehyde (MDA), and chlorophyll in tea leaves after treatment with different F concentrations. In addition, we focused on the content of organic acids, the gene transcription of malate dehydrogenase (MDH), glycolate oxidase (GO) and citrate synthase (CS) and the relative enzyme activity involved in the tolerance to F in C. sinensis. We also examined the role of Al in this process by analyzing the content of these physiological indicators in tea leaves treated with F and Al. Our results demonstrate that increased MDA content, together with decreased chlorophyll content and soluble proteins are responsible for oxidative damage and metabolism inhibition at high F concentration. Moreover, increased antioxidant enzymes activity regulates ROS damage to protect tea leaves during F stress. Furthermore, exogenous Al alleviated F stress in tea leaves through the regulation of MDA content and antioxidant enzymes activity. In addition, organic acids in exudate stimulated root growth in tea plants exposed to low F concentrations are regulated by MDH, GO, and CS. In addition, Al can stimulate the exudation of organic acids, and may participate in regulating rhizosphere pH of the roots through the interaction with F, eventually leading to the response to F stress in C. sinensis.

Physiological and iTRAQ-based proteomic analyses reveal the function of exogenous γ-aminobutyric acid (GABA) in improving tea plant (Camellia sinensis L.) tolerance at cold temperature.

BACKGROUND: Internal γ-Aminobutyric Acid (GABA) interacting with stress response substances may be involved in the regulation of differentially abundant proteins (DAPs) associated with optimum temperature and cold stress in tea plants (Camellia sinensis (L.) O. Kuntze). RESULTS: Tea plants supplied with or without 5.0 mM GABA were subjected to optimum or cold temperatures in this study. The increased GABA level induced by exogenous GABA altered levels of stress response substances - such as glutamate, polyamines and anthocyanins - in association with improved cold tolerance. Isobaric tags for relative and absolute quantification (iTRAQ) - based DAPs were found for protein metabolism and nucleotide metabolism, energy, amino acid transport and metabolism other biological processes, inorganic ion transport and metabolism, lipid metabolism, carbohydrate transport and metabolism, biosynthesis of secondary metabolites, antioxidant and stress defense. CONCLUSIONS: The iTRAQ analysis could explain the GABA-induced physiological effects associated with cold tolerance in tea plants. Analysis of functional protein-protein networks further showed that alteration of endogenous GABA and stress response substances induced interactions among photosynthesis, amino acid biosynthesis, and carbon and nitrogen metabolism, and the corresponding differences could contribute to improved cold tolerance of tea plants.

Overexpression of an ABA-dependent grapevine bZIP transcription factor, VvABF2, enhances osmotic stress in Arabidopsis.

KEY MESSAGE: Overexpression of grapevine VvABF2 gene could enhance osmotic stress tolerance in Arabidopsis thaliana but fully required for ABA signaling. The abscisic acid (ABA)-dependent AREB/ABF-SnRK2 pathway has been demonstrated to play a pivotal role in response to osmotic stress in model plants. However, its function in other specific species, for example grapevine, has not been fully characterized. In this study, grapevine (Vitis vinifera L.) ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 (VvABF2), a homologous gene of AREB/ABFs form Arabidopsis, was isolated and constitutively expressed in Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. The VvABF2 transgenic Arabidopsis plants showed to be more sensitive to exogenous ABA compared to wild type plants and exhibited significant osmotic tolerance, like polyethylene glycol (PEG) and drought but fully required ABA for signaling. This fact was further confirmed by its downstream gene expression assays. In addition, the determination of ROS antioxidant enzymes (including SOD, POD and CAT) and the MDA of transgenic lines indicated that overexpression of VvABF2 in Arabidopsis significantly increased ROS scavenging ability and thereby reduced the cell membrane damage, which might be ABA-independent. Our results provide evidence that VvABF2 has a similar function to the Arabidopsis homolog in response to osmotic stresses, and that there is a similar ancestral function of this gene in ABA-dependent response to stresses in grapevine.

Transcriptome Analysis Reveals the Mechanism of Fluoride Treatment Affecting Biochemical Components in Camellia sinensis.

Tea (Camellia sinensis (L.) O. Kuntze), one of the main crops in China, is high in various bioactive compounds including flavonoids, catechins, caffeine, theanine, and other amino acids. C. sinensis is also known as an accumulator of fluoride (F), and the bioactive compounds are affected by F, however, the mechanism remains unclear. Here, the effects of F treatment on the accumulation of F and major bioactive compounds and gene expression were investigated, revealing the molecular mechanisms affecting the accumulation of bioactive compounds by F treatment. The results showed that F accumulation in tea leaves gradually increased under exogenous F treatments. Similarly, the flavonoid content also increased in the F treatment. In contrast, the polyphenol content, free amino acids, and the total catechins decreased significantly. Special amino acids, such as sulfur-containing amino acids and proline, had the opposite trend of free amino acids. Caffeine was obviously induced by exogenous F, while the theanine content peaked after two day-treatment. These results suggest that the F accumulation and content of bioactive compounds were dramatically affected by F treatment. Furthermore, differentially expressed genes (DEGs) related to the metabolism of main bioactive compounds and amino acids, especially the pivotal regulatory genes of catechins, caffeine, and theanine biosynthesis pathways, were identified and analyzed using high-throughput Illumina RNA-Seq technology and qRT-PCR. The expression of pivotal regulatory genes is consistent with the changes of the main bioactive compounds in C. sinensis leaves, indicating a complicated molecular mechanism for the above findings. Overall, these data provide a reference for exploring the possible molecular mechanism of the accumulation of major bioactive components such as flavonoid, catechins, caffeine, theanine and other amino acids in tea leaves in response to fluoride treatment.

Increased salt tolerance with overexpression of cation/proton antiporter 1 genes: a meta-analysis.

Cation/proton antiporter 1 (CPA1) genes encode cellular Na+ /H+ exchanger proteins, which act to adjust ionic balance. Overexpression of CPA1s can improve plant performance under salt stress. However, the diversified roles of the CPA1 family and the various parameters used in evaluating transgenic plants over-expressing CPA1s make it challenging to assess the complex functions of CPA1s and their physiological mechanisms in salt tolerance. Using meta-analysis, we determined how overexpression of CPA1s has influenced several plant characteristics involved in response and resilience to NaCl stress. We also evaluated experimental variables that favour or reduce CPA1 effects in transgenic plants. Viewed across studies, overexpression of CPA1s has increased the magnitude of 10 of the 19 plant characteristics examined, by 25% or more. Among the ten moderating variables, several had substantial impacts on the extent of CPA1 influence: type of culture media, donor and recipient type and genus, and gene family. Genes from monocotyledonous plants stimulated root K+ , root K+ /Na+ , total chlorophyll, total dry weight and root length much more than genes from dicotyledonous species. Genes transformed to or from Arabidopsis have led to smaller CPA1-induced increases in plant characteristics than genes transferred to or from other genera. Heterogeneous expression of CPA1s led to greater increases in leaf chlorophyll and root length than homologous expression. These findings should help guide future investigations into the function of CPA1s in plant salt tolerance and the use of genetic engineering for breeding of resistance.

Genome-wide identification and analysis of mitogen activated protein kinase kinase kinase gene family in grapevine (Vitis vinifera).

BACKGROUND: Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize. RESULTS: In this study, we performed a genome-wide survey and identified 45 MAPKKK genes in the grapevine genome. Chromosome location, phylogeny, gene structure and conserved protein motifs of MAPKKK family in grapevine have been analyzed to support the prediction of these genes. In the phylogenetic analysis, MAPKKK genes of grapevine have been classified into three subgroups as described for Arabidopsis, named MEKK, ZIK and RAF, also confirmed in grapevine by the analysis of conserved motifs and exon-intron organizations. By analyzing expression profiles of MAPKKK genes in grapevine microarray databases, we highlighted the modulation of different MAPKKKs in different organs and distinct developmental stages. Furthermore, we experimentally investigated the expression profiles of 45 grape MAPKKK genes in response to biotic (powdery mildew) and abiotic stress (drought), as well as to hormone (salicylic acid, ethylene) and hydrogen peroxide treatments, and identified several candidate MAPKKK genes that might play an important role in biotic and abiotic responses in grapevine, for further functional characterization. CONCLUSIONS: This is the first comprehensive experimental survey of the grapevine MAPKKK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses, and the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPK cascade in grapevine.

Causality orientations and spontaneous mental contrasting.

Mental contrasting is a motivational behavior change strategy necessary for strong goal commitment. Meanwhile, general causality orientations are motivational patterns that represent individuals' motivation for behavior change and the reason for their goal commitment. The current study explored whether causality orientations predict spontaneous mental contrasting in Chinese university students. Study 1 investigated whether academic autonomy, control, and amotivated orientations correlate with spontaneous mental contrasting about an important academic goal. The findings of Study 1 reveal that autonomy orientation did not correlate with mental contrasting, whereas control and amotivated orientations were negatively correlated with mental contrasting. Study 2 investigated whether priming autonomy and control orientations, in addition to the neutral condition, would induce spontaneous mental contrasting about an academic goal related to the students' research topic. The results of Study 2 revealed that the autonomy condition orientation did not differ significantly from the controlled orientation condition. However, when compared to the neutral condition, the autonomy condition significantly predicted mental contrasting, whereas the controlled orientation condition did not show any significant difference. In Study 2, the autonomy-oriented participants generated more spontaneous mental contrast than the control orientation and neutral conditions. The findings show that controlled and amotivated orientations predicted negative mental contrasting. As a result, controlled and amotivated students must learn how to use mental contrasting to achieve high levels of goal commitment and achievement. Lastly, the study discussed its implications, limitations, and suggestions for future research.

Intercropping fruit trees in tea plantation improves soil properties and the formation of tea quality components.

Intercropping has been recommended as a beneficial cropping practice for improving soil characteristic and tea quality. However, there is limited research on the effects of intercropping fruit trees on soil chemical properties, soil aggregate structure, and tea quality components. In this study, intercropping fruit trees, specifically loquats and citrus, had a significant impact on the total available nutrients, AMN, and AP in soil. During spring and autumn seasons, the soil large-macroaggregates (>2 mm) proportion increased by 5.93% and 19.03%, as well as 29.23% and 19.14%, respectively, when intercropping loquats and citrus. Similarly, intercropping waxberry resulted in a highest small-macroaggregates (0.25 mm-2 mm) proportion at 54.89% and 77.32%. Soil aggregate stability parameters of the R0.25, MWD, and GMD were generally considered better soil aggregate stability indicators, and significantly improved in intercropping systems. Intercropping waxberry with higher values for those aggregate stability parameters and lower D values, showed a better soil aggregate distribution, while intercropping loquats and citrus at higher levels of AMN and AP in different soil aggregate sizes. As the soil aggregate sizes increased, the AMN and AP contents gradually decreased. Furthermore, the enhanced levels of amino acids were observed under loquat, waxberry, and citrus intercropping in spring, which increased by 27.98%, 27.35%, and 26.21%, respectively. The contents of tea polyphenol and caffeine were lower under loquat and citrus intercropping in spring. These findings indicated that intercropping fruit trees, specifically loquat and citrus, have immense potential in promoting the green and sustainable development of tea plantations.

Leguminous green manure intercropping changes the soil microbial community and increases soil nutrients and key quality components of tea leaves.

Intercropping, a green and sustainable planting pattern, has demonstrated positive effects on plant growth and the soil environment. However, there is currently little research on the influence of intercropping leguminous plants and using them as green manure on the soil environment and tea quality. During the profuse flowering period of Chinese milkvetch, the contents of tea amino acids and soluble sugar in intercropping tea plants with soybean increased by 6.89 and 54.58%. Moreover, there was 27.42% increase in soil ammonium nitrogen and 21.63% increase in available nitrogen. When Chinese milkvetch was returned to soil for 1 month during its profuse flowering period, the soybean and Chinese milkvetch as green manure enhanced tea amino acids and soluble sugar by 9.11 and 33.96%, and soil ammonium nitrogen, nitrate nitrogen and available nitrogen increased by 25.04, 77.84, and 48.90%. Intercropping systems also have positive effects on tea quality components, soil fertility, and soil microbial communities during the profuse flowering period of soybeans and when soybeans with this period were returned to the field for 1 month. Furthermore, the soil fertility index was significantly increased, especially in the intercropping system of tea-soybean-Chinese milkvetch. The soil bacterial community complexity and fungal community interactions were significantly increased. Soil pH, nitrate nitrogen, and available phosphorus were found to be crucial influencing factors on soil microbial communities, specifically bacterial communities. These results highlight the significance of optimizing intercropping systems to improve the soil environment and tea quality components. They also provide a theoretical foundation for promoting the sustainable development of tea plantations.

Minigene Assay as an Effective Molecular Diagnostic Strategy in Determining the Pathogenicity of Noncanonical Splice-Site Variants in FLCN.

Primary spontaneous pneumothorax (PSP) or pulmonary cyst is one of the manifestations of Birt-Hogg-Dubé syndrome, which is caused by pathogenic variants in FLCN gene. Genetic testing in patients with PSP identifies a certain number of missense or intronic variants. These variants are usually considered as variants of uncertain significance, whose functional interpretations pose a challenge in clinical genetics. To improve recognition of pathogenic splice-altering variants in FLCN gene, computational tools are used to prioritize potential splice-altering variants and then a hybrid minigene assay is performed to verify the RNA splicing pattern. Herein, variants in FLCN exon 11 and its flanking sequence are focused. Eight variants detected in 11 patients with PSP are evaluated, and six variants are prioritized by in silico tools as potential splice-altering variants of uncertain significance. Four variants (c.1177-5_1177-3delCTC, c.1292_1300+4del, c.1300+4C>T, and c.1300+5G>A) are demonstrated by minigene assay to alter RNA splicing of FLCN, and the last three of them are novel. RT-PCR of patient-derived RNA gives consistent results. Genotype-phenotype correlation analysis in patients with PSP with these variants demonstrates good concordance. Our results underline the importance of RNA analysis, which could provide molecular evidence for pathogenicity of a variant, and provide essential information for the clinical interpretation of variants. Combining the clinical information, a definitive diagnosis could be made.

Genome-Wide Association Studies of Biluochun Tea Plant Populations in Dongting Mountain and Comprehensive Identification of Candidate Genes Associated with Core Agronomic Traits by Four Analysis Models.

The elite germplasm resources are key to the beautiful appearance and pleasant flavor of Biluochun tea. We collected and measured the agronomic traits of 95 tea plants to reveal the trait diversity and breeding value of Biluochun tea plant populations. The results revealed that the agronomic traits of Biluochun tea plant populations were diverse and had high breeding value. Additionally, we resequenced these tea plant populations to reveal genetic diversity, population structure, and selection pressure. The Biluochun tea plant populations contained two groups and were least affected by natural selection based on the results of population structure and selection pressure. More importantly, four non-synonymous single nucleotide polymorphisms (nsSNPs) and candidate genes associated with (-)-gallocatechin gallate (GCG), (-)-gallocatechin (GC), and caffeine (CAF) were detected using at least two GWAS models. The results will promote the development and application of molecular markers and the utilization of elite germplasm from Biluochun populations.

Development of Novel Lipid-Based Formulations for Water-Soluble Vitamin C versus Fat-Soluble Vitamin D3.

The aim of this study was to develop a facile and novel lipid-based formulation of vitamin C and vitamin D3. Liposomes loaded with vitamin C and D3 were characterized using transmission electron microscopy (TEM) and zeta potential measurements for evaluating morphology, particle size and physical stability. HPLC was employed to quantify the content of vitamin C and vitamin D3 in their liposomal forms. The UHPLC analysis of the lipid-based vitamin formulation is an easy and rapid method for the characterization as well as the quantification of all components. In addition, encapsulation efficiency, vitamin loading and stability analysis were performed by the UHPLC method, in order to evaluate the reliability of the optimized lipid-based formulation. The TEM results provided key support for the core type of liposome structure in the formulations, whereas the HPLC results indicated that the liposomal vitamin C and D3 systems were homogeneous, and did not undergo phase separation. Taken together, the results demonstrate that liposomal encapsulated vitamins (vitamin C and D3) possess a unilamellar vesicle morphology with uniform particle size, despite differences in the hydrophile-lipophile profiles of the vitamins. The highly efficient encapsulation properties of such liposomal constructs are proposed to contribute to enhanced vitamin bioavailability.