(Z, Z, Z)-3,6,9-Nonadecadiene, a potential inhibitor of sex pheromone of Grey Tea Geometrid (Lepidoptera: Geometridae): electroantennogram test, wind tunnel, and in silico study.

Sex pheromone analogs have high structural similarity to sex pheromone components. They also play a role in studying many agricultural pests. In our study, (Z, Z, Z)-3,6,9-nonadecadiene (Z3Z6Z9-19:Hy) was successfully synthesized, which is an analogue to 1 of 2 sex pheromone components of Ectropis grisescens Warren (Z, Z, Z)-3,6,9-octadecatriene (Z3Z6Z9-18:Hy), and it showed potential inhibition in experiments. In the electroantennogram test, Z3Z6Z9-19:Hy showed a dose-dependent response, and only measured half the response of Z3Z9-6,7-epo-18:Hy. However, the compound significantly reduced positive response of E. grisescens males by up to 70% in the Y-tube olfactometer. Furthermore, in the wind tunnel, it significantly inhibited all types of behavioral responses. The percentage of moths contacting the pheromone odor source was reduced even at the lowest dose tested. In silico study afterward, molecular docking results showed affinity between Z3Z6Z9-19:Hy and sensory neuron membrane protein 1. Our study revealed the potential of Z3Z6Z9-19:Hy as a sex pheromone inhibitor, which would provide new tools for monitoring and mating disruption of E. grisescens.

Clinicopathologic features and abnormal signaling pathways in plasmablastic lymphoma: a multicenter study in China.

BACKGROUND: Plasmablastic lymphoma (PBL) is a rare but aggressive B-cell lymphoma subtype with poor prognosis. Knowledge about the etiology, clinicopathologic and molecular features, and outcomes of PBL is limited. This study aimed to examine the clinicopathologic characteristics, therapeutic approaches, and clinical outcomes of PBL patients in a Chinese population. METHODS: A total of 102 PBL patients were recruited from three cancer centers. The pathologic features and clinical outcomes of 56 patients with available treatment details and follow-up data were reviewed and analyzed. RNA sequencing was performed in 6 PBL and 11 diffuse large B-cell lymphoma (DLBCL) patients. RESULTS: Most patients in our cohort were male (n = 36, 64.3%), and 35 patients presented with Ann Arbor stage I/II disease at diagnosis. All these patients showed negative findings for human immunodeficiency virus, and the vast majority of patients in our cohort were immunocompetent. Lymph nodes (n = 13, 23.2%) and gastrointestinal tract (n = 10, 17.9%) were the most commonly involved site at presentation. Post-treatment complete remission (CR) was the only prognostic factor affecting overall survival (OS) and progression-free survival (PFS) in the multivariate analysis. RNA-seq demonstrated that B-cell receptor (BCR), T-cell receptor (TCR), P53, calcium signaling, and Wnt signaling pathways were significantly downregulated in PBLs compared with GCB (or non-GCB) DLBCLs. CONCLUSIONS: In this multicenter study in the Chinese population, PBL mainly occurred in immunocompetent individuals and most patients present with early-stage disease at diagnosis. Post-treatment CR was an important prognostic factor affecting OS and PFS. RNA-seq showed that the B-cell receptor (BCR), P53, calcium signaling, cell adhesion molecules, and Wnt signaling pathways significantly differed between PBL and GCB (or non-GCB) DLBCL, which provided theoretical basis for its pathogenesis and future treatment.

Microbial and Nonvolatile Chemical Diversities of Chinese Dark Teas Are Differed by Latitude and Pile Fermentation.

Understanding the microbial and chemical diversities, as well as what affects these diversities, is important for modern manufacturing of traditional fermented foods. In this work, Chinese dark teas (CDTs) that are traditional microbial fermented beverages with relatively high sample diversity were collected. Microbial DNA amplicon sequencing and mass spectrometry-based untargeted metabolomics show that the CDT microbial ß diversity, as well as the nonvolatile chemical α and ß diversities, is determined by the primary impact factors of geography and manufacturing procedures, in particular, latitude and pile fermentation after blending. A large number of metabolites sharing between CDTs and fungi were discovered by Feature-based Molecular Networking (FBMN) on the Global Natural Products Social Molecular Networking (GNPS) web platform. These molecules, such as prenylated cyclic dipeptides and B-vitamins, are functionally important for nutrition, biofunctions, and flavor. Molecular networking has revealed patterns in metabolite profiles on a chemical family level in addition to individual structures.

Preferentially expressed antigen in melanoma immunohistochemistry as an adjunct for differential diagnosis in acral lentiginous melanoma and acral nevi.

Preferentially expressed antigen in melanoma (PRAME) has shown promising utility in distinguishing benign melanocytic lesions from melanomas, but knowledge of its expression pattern in acral lentiginous melanoma (ALM) and acral nevi (ANs) is limited. Immunohistochemical expression of PRAME was examined in 75 ALMs and 34 ANs. The clinical and histopathologic characteristics of patients with ALM were collected. PRAME was immunoreactive in 89.3% (67/75) of ALMs, but entirely negative in 94.1% (32/34) of ANs. When staining at least 50% of lesional melanocytes was determined as positivity, the sensitivity and specificity of PRAME for distinguishing ALM from ANs were 69.3% and 100%, respectively. Seventy-one cases of ALMs had tumor cells in the epidermis; 71.8% (51/71) of them showed positive for PRAME. By contrast, 61 ALMs had tumor cells in the dermis; 65.6% (40/61) exhibited positive expression. Twenty-nine of 39 (74.4%) epithelioid cell ALMs were observed to be positive for PRAME. By comparison, 63.8% (23/36) of ALMs with spindle tumor cells were positive for PRAME. However, PRAME positive expression was not associated with any clinical and histopathologic characteristics of patients with ALM, including Breslow thickness, ulcer, cytomorphology, lymph node metastasis, or tumor-infiltrated lymphocytes (TILs). Nevertheless, we observed that 82.6% (19/23) of ALMs with lymph node involvement at diagnosis expressed PRAME, compared with 57.6% (20/35) of those without. In summary, PRAME immunohistochemistry can serve as a helpful adjunct in the differential diagnosis of ALMs and ANs with good sensitivity and high specificity. Additionally, PRAME tends to have a higher positive rate in epidermal melanocytes than in the dermis and is inclined to express in epithelioid cells than in spindle cells of ALMs.

Untargeted Metabolomics Combined with Bioassay Reveals the Change in Critical Bioactive Compounds during the Processing of Qingzhuan Tea.

Qingzhuan tea (QZT) is a typical Chinese dark tea that has a long-time manufacturing process. In the present study, liquid chromatography coupled with tandem mass spectrometry was used to study the chemical changes of tea samples during QZT processing. Untargeted metabolomics analysis revealed that the pile-fermentation and turnover (post-fermentation, FT) was the crucial stage in transforming the main compounds of QZT, whose contents of flavan-3-ols and flavonoids glycosides were decreased significantly. The bioactivities, including the antioxidant capacities and inhibitory effects on α-amylase and α-glucosidase, were also reduced after the FT process. It was suggested that although the QZT sensory properties improved following pile-fermentation and aging, the bioactivities remained restrained. Correlation analysis indicated that the main galloylated catechins and flavonoid glycosides were highly related to their antioxidant capacity and inhibitory effects on α-amylase and α-glucosidase.

Universal extracellular vesicles and PD-L1+ extracellular vesicles detected by single molecule array technology as circulating biomarkers for diffuse large B cell lymphoma.

Plasma extracellular vesicles (EVs) have been reported to be a promising source of diagnostic and prognostic biomarkers in various cancers. However, further research in this area is needed due to the limitations of circulating extracellular vesicles detection methods. Using the Single Molecule array (SiMoa) technology, we developed two extracellular vesicle detection assays, CD9-CD63 and PD-L1-CD63, to determine circulating universal EVs and PD-L1 positive EVs, respectively. A total of 164 diffuse large B-cell lymphoma (DLBCL) patients were retrospectively included in this study. Compared with healthy volunteers (n = 25), elevated CD9-CD63 and PD-L1-CD63 signals were detected in the plasma of DLBCL patients (n = 164). High CD9-CD63 signals was associated with molecular subtype, extranodal site and treatment response in DLBCL. A high PD-L1-CD63 signal was also associated with certain clinical features, including extranodal site and treatment response. CD9-CD63 and PD-L1-CD63 signals were found to be important prognostic factors for both progression-free and overall survival. Furthermore, PD-L1-positive EVs were found in all patients, though PD-L1 protein expression was positive in only 35.4% (17/48) of tumor biopsies. No correlation was found between circulating PD-L1+ EVs and soluble PD-L1 (sPD-L1) levels. Our results show that plasma universal EV and PD-L1-positive EV levels are significantly elevated in DLBCL and might serve as biomarkers for predicting survival outcomes in DLBCL patients.

Efficient isolation and purification of tissue-specific protoplasts from tea plants (Camellia sinensis (L.) O. Kuntze).

BACKGROUND: Plant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis. An effective and sustainable method for preparing protoplasts from tea plants has yet to be established. The protoplasts were osmotically isolated, and the isolation and purification procedures were optimized. Various potential factors affecting protoplast preparation, including enzymatic composition and type, enzymatic hydrolysis duration, mannitol concentration in the enzyme solution, and iodixanol concentration, were evaluated. RESULTS: The optimal conditions were 1.5% (w/v) cellulase and 0.4-0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. The highest protoplast yield was 3.27 × 106 protoplasts g-1 fresh weight. As determined through fluorescein diacetate staining, maximal cell viability was 92.94%. The isolated protoplasts were round and regularly shaped without agglomeration, and they were less than 20 µm in diameter. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were also observed. CONCLUSIONS: In summary, we reported on a simple, efficient method for preparing protoplasts of whole-organ tissue from tea plant. The findings are expected to contribute to the rapid development of tea plant biology.

Fluoride absorption, transportation and tolerance mechanism in Camellia sinensis, and its bioavailability and health risk assessment: a systematic review.

Tea is the one of the most popular non-alcoholic caffeinated beverages in the world. Tea is produced from the tea plant (Camellia sinensis (L.) O. Kuntze), which is known to accumulate fluoride. This article systematically analyzes the literature concerning fluoride absorption, transportation and fluoride tolerance mechanisms in tea plants. Fluoride bioavailability and exposure levels in tea infusions are also reviewed. The circulation of fluoride within the tea plantation ecosystems is in a positive equilibrium, with greater amounts of fluoride introduced to tea orchards than removed. Water extractable fluoride and magnesium chloride (MgCl2 ) extractable fluoride in plantation soil are the main sources of absorption by tea plant root via active trans-membrane transport and anion channels. Most fluoride is readily transported through the xylem as F- /F-Al complexes to leaf cell walls and vacuole. The findings indicate that tea plants employ cell wall accumulation, vacuole compartmentalization, and F-Al complexes to co-detoxify fluoride and aluminum, a possible tolerance mechanism through which tea tolerates higher levels of fluoride than most plants. Furthermore, dietary and endogenous factors influence fluoride bioavailability and should be considered when exposure levels of fluoride in commercially available dried tea leaves are interpreted. The relevant current challenges and future perspectives are also discussed. © 2020 Society of Chemical Industry.

Effects of umbilical cord mesenchymal stem cells loaded with graphene oxide granular lubrication on cytokine levels in animal models of knee osteoarthritis.

BACKGROUND: The aim of this study was to use umbilical cord mesenchymal stem cells (UCMSCs) loaded with graphene oxide (GO) granular lubricant to treat knee osteoarthritis (KOA) animal models and to analyze their effect on cytokine levels in the articular cavity. METHODS: Twenty-four New Zealand rabbit models of KOA were established by the modified Hulth and cartilage injury method, and they were assigned to the blank group, the GO group, the UCMSC group, and the GO + UCMSC group, each group containing six animal models. The GO and UCMSC groups were treated by a single intra-articular injection. The treatment was started one month after surgical modeling, and the observation period was eight weeks. The expression levels of nitric oxide (NO), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), glycosaminoglycan (GAG), and collagen-II (COL-II) in serum and articular fluid after treatment were compared to analyze the efficacy. RESULTS: The GO granular lubricant caused no significant improvement in the intra-articular environment of the knee joint, and UCMSCs caused a certain degree of improvement in the inflammatory environment. The improvement results of NO, IL-6, TNF-α, GAG, and COL-II were the best in the GO + UCMSC group, but the improvement results of inflammatory cytokine levels in serum and articular fluid were not consistent, especially the differences in NO, IL-6, and TNF-α were greater. CONCLUSION: UCMSCs loaded with the GO granular lubricant can reduce the inflammatory level and improve the level of biochemical environment in the articular cavity, and thus promote cartilage repair.

Metabolics and ionomics responses of tea leaves (Camellia sinensis (L.) O. Kuntze) to fluoride stress.

Tea plant (Camellia sinensis (L.) O. Kuntze) is known to accumulate high concentrations of fluoride (F) in its leaves; however, the underlying mechanism of F accumulation remains unclear. The main objective of this study was to investigate the homeostatic self-defense mechanisms of tea leaves to F supplementation (0, 5, 20, and 50 mgL-1) by metabolomics and ionomics. We identified a total of 96 up-regulated and 40 down-regulated metabolites in tea leaves treated with F. Of these different compounds, minor polypeptides, carbohydrates and amino acids played valuable roles in the F-tolerating mechanism of tea plant. After F treatments, the concentrations of sodium (Na), ferrum (Fe), manganese (Mn), and molybdenum (Mo) were significantly increased in tea leaves, whereas the aluminum (Al) was decreased. These findings suggest that the ionic balance and metabolites are attributable to the development of F tolerance, providing new insight into tea plant adaptation to F stress.

Feature-Based Molecular Networking Analysis of the Metabolites Produced by In Vitro Solid-State Fermentation Reveals Pathways for the Bioconversion of Epigallocatechin Gallate.

Dark teas are prepared by a microbial fermentation process. Flavan-3-ol B-ring fission analogues (FBRFAs) are some of the key bioactive constituents that characterize dark teas. The precursors and the synthetic mechanism involved in the formation of FBRFAs are not known. Using a unique solid-state fermentation system with ß-cyclodextrin inclusion complexation as well as targeted chromatographic isolation, spectroscopic identification, and Feature-based Molecular Networking on the Global Natural Products Social Molecular Networking web platform, we reveal that dihydromyricetin and the FBRFAs, including teadenol A and fuzhuanin A, are derived from epigallocatechin gallate upon exposure to fungal strains isolated from Fuzhuan brick tea. In particular, the strains from subphylum Pezizomycotina were key drivers for these B-/C-ring oxidation transformations. These are the same transformations seen during the fermentation process of dark teas. These discoveries set the stage to enrich dark teas and other food products for these health-promoting constituents.

Genome-Wide Analysis of Serine Carboxypeptidase-Like Acyltransferase Gene Family for Evolution and Characterization of Enzymes Involved in the Biosynthesis of Galloylated Catechins in the Tea Plant (Camellia sinensis).

Tea (Camellia sinensis L.) leaves synthesize and concentrate a vast array of galloylated catechins (e.g., EGCG and ECG) and non-galloylated catechins (e.g., EGC, catechin, and epicatechin), together constituting 8%-24% of the dry leaf mass. Galloylated catechins account for a major portion of soluble catechins in tea leaves (up to 75%) and make a major contribution to the astringency and bitter taste of the green tea, and their pharmacological activity for human health. However, the catechin galloylation mechanism in tea plants is largely unknown at molecular levels. Previous studies indicated that glucosyltransferases and serine carboxypeptidase-like acyltransferases (SCPL) might be involved in the process. However, details about the roles of SCPLs in the biosynthesis of galloylated catechins remain to be elucidated. Here, we performed the genome-wide identification of SCPL genes in the tea plant genome. Several SCPLs were grouped into clade IA, which encompasses previously characterized SCPL-IA enzymes with an acylation function. Twenty-eight tea genes in this clade were differentially expressed in young leaves and vegetative buds. We characterized three SCPL-IA enzymes (CsSCPL11-IA, CsSCPL13-IA, CsSCPL14-IA) with galloylation activity toward epicatechins using recombinant enzymes. Not only the expression levels of these SCPLIA genes coincide with the accumulation of galloylated catechins in tea plants, but their recombinant enzymes also displayed ß-glucogallin:catechin galloyl acyltransferase activity. These findings provide the first insights into the identities of genes encoding glucogallin:catechin galloyl acyltransferases with an active role in the biosynthesis of galloylated catechins in tea plants.

Positron-emitting tracer imaging of fluoride transport and distribution in tea plant.

BACKGROUND: Tea (Camellia sinensis (L.) O. Kuntze) is a hyper-accumulator of fluoride (F). To understand F uptake and distribution in living plants, we visually evaluated the real-time transport of F absorbed by roots and leaves using a positron-emitting (18 F) fluoride tracer and a positron-emitting tracer imaging system. RESULTS: F arrived at an aerial plant part about 1.5 h after absorption by roots, suggesting that tea roots had a retention effect on F, and then was transported upward mainly via the xylem and little via the phloem along the tea stem, but no F was observed in the leaves within the initial 8 h. F absorbed via a cut petiole (leaf 4) was mainly transported downward along the stem within the initial 2 h. Although F was first detected in the top and ipsilateral leaves, it was not detected in tea roots by the end of the monitoring. During the monitoring time, F principally accumulated in the node. CONCLUSION: F uptake by the petiole of excised leaf and root system was realized in different ways. The nodes indicated that they may play pivotal roles in the transport of F in tea plants. © 2020 Society of Chemical Industry.

Tea plant genomics: achievements, challenges and perspectives.

Tea is among the world's most widely consumed non-alcoholic beverages and possesses enormous economic, health, and cultural values. It is produced from the cured leaves of tea plants, which are important evergreen crops globally cultivated in over 50 countries. Along with recent innovations and advances in biotechnologies, great progress in tea plant genomics and genetics has been achieved, which has facilitated our understanding of the molecular mechanisms of tea quality and the evolution of the tea plant genome. In this review, we briefly summarize the achievements of the past two decades, which primarily include diverse genome and transcriptome sequencing projects, gene discovery and regulation studies, investigation of the epigenetics and noncoding RNAs, origin and domestication, phylogenetics and germplasm utilization of tea plant as well as newly developed tools/platforms. We also present perspectives and possible challenges for future functional genomic studies that will contribute to the acceleration of breeding programs in tea plants.

Evaluation of the feasibility of short-term electrodialysis for separating naturally occurring fluoride from instant brick tea infusion.

BACKGROUND: Removing excessive naturally occurring fluoride from tea and/or infusions is difficult because the process has low efficiency and causes secondary pollution. In this study, a novel electrodialysis (ED) technology was developed. We examined the effect of crucial parameters (electrolyte concentration, operation voltage, ED duration and initial concentration of the tea infusion) on defluoridation performance using a highly efficient ion-exchange membrane with five-compartment cells. RESULTS: The most effective ED system results were obtained at an electrolyte concentration of 10 g kg-1 and operating voltage of 20 V. Moreover, the fluoride removal capacity (10.70-66.93%) was highly dependent on the ED duration (1-15 min) and initial concentration of the tea infusion (0.5-10 g kg-1 ). The longer the ED duration and the lower the initial concentration, the higher was the defluoridation performance. During ED, limited loss of the main inclusions (total polyphenols, catechins, caffeine and selected ions) was observed. Furthermore, the D201 anion resin-filled ED stack (0.5-5 g) and improvement of concentrate compartment electrolyte (≥5 times the dilute compartment electrolyte) in the ED system enhanced the defluoridation rate significantly. CONCLUSION: ED is a potentially effective method that can be used for defluoridation in the deep processing of tea products. © 2019 Society of Chemical Industry.

Enantiomeric Trimethylallantoin Monomers, Dimers, and Trimethyltriuret: Evidence for an Alternative Catabolic Pathway of Caffeine in Tea Plant.

Racemic trimethylallantoin monomer (1), mesomeric and racemic trimethylallantoin dimers (2 and 3), were isolated from tea. Two pairs of optically pure enantiomers (1a, 1b and 3a, 3b) were separated by chiral column from the two racemes (1 and 3). Their structures were elucidated by a combination of extensive spectroscopic techniques, single-crystal X-ray diffraction, and experimental and calculated electronic circular dichroism. A novel caffeine catabolic pathway was proposed based on the caffeine stable isotopic tracer experiments.

Tea Plant Information Archive: a comprehensive genomics and bioinformatics platform for tea plant.

Tea is the world's widely consumed nonalcohol beverage with essential economic and health benefits. Confronted with the increasing large-scale omics-data set particularly the genome sequence released in tea plant, the construction of a comprehensive knowledgebase is urgently needed to facilitate the utilization of these data sets towards molecular breeding. We hereby present the first integrative and specially designed web-accessible database, Tea Plant Information Archive (TPIA; http://tpia.teaplant.org). The current release of TPIA employs the comprehensively annotated tea plant genome as framework and incorporates with abundant well-organized transcriptomes, gene expressions (across species, tissues and stresses), orthologs and characteristic metabolites determining tea quality. It also hosts massive transcription factors, polymorphic simple sequence repeats, single nucleotide polymorphisms, correlations, manually curated functional genes and globally collected germplasm information. A variety of versatile analytic tools (e.g. JBrowse, blast, enrichment analysis, etc.) are established helping users to perform further comparative, evolutionary and functional analysis. We show a case application of TPIA that provides novel and interesting insights into the phytochemical content variation of section Thea of genus Camellia under a well-resolved phylogenetic framework. The constructed knowledgebase of tea plant will serve as a central gateway for global tea community to better understand the tea plant biology that largely benefits the whole tea industry.

Using stable isotope signatures to delineate the geographic point-of-origin of Keemun black tea.

BACKGROUND: Confirmation of food labeling that claims production in a small geographic region is critical to traceability, quality control and brand protection. In the current study, isotope ratio mass spectrometry (IRMS) was used to generate profiles of δ13 C and δ15 N to determine if the stable isotope signatures of Keemun black tea differ within the three counties that claim production. Other factors (cultivar type, leaf maturity and manufacturing process) were considered for their potential effects. RESULTS: Both cultivar type and leaf maturity have remarkable impact on the δ15 N values of tea leaves, and that the cultivar influenced the δ13 C values. Keemun black tea from Qimen county could be easily discriminated from samples from Dongzhi and Guichi counties based on δ15 N signatures. The k-NN model was cross-validated with an accuracy of 91.6%. Environmental factors and/or genotype seem to be the major reasons for δ15 N differences in Keemun black tea from the selected regions. CONCLUSION: This article provides a potential effective method to delineate the geographic point-of-origin of Keemun black tea based on δ15 N signatures. © 2018 Society of Chemical Industry.

Flavoalkaloids with a Pyrrolidinone Ring from Chinese Ancient Cultivated Tea Xi-Gui.

Chinese Xi-Gui tea is one ancient cultivated variety of Camellia sinensis var. assamica. At present, it is used for producing expensive and elite tea in China. Five new flavoalkaloids, (-)-6-(5''' S)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (ester-type catechins pyrrolidinone E, etc-pyrrolidinone E, 1), (-)-6-(5''' R)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone F, 2) (-)-8-(5''' S)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone G, 3a), (-)-8-(5''' S)- N-ethyl-2-pyrrolidinone-catechin-3- O-gallate (etc-pyrrolidinone I, 4a), (-)-8-(5''' R)- N-ethyl-2-pyrrolidinone-catechin-3- O-gallate (etc-pyrrolidinone J, 4b), and one new naturally occurring natural product (-)-8-(5''' R)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone H, 3b) together with the known flavoalkaloids etc-pyrrolidinones A-D (5, 6, 7a, and 7b) were detected and isolated from Xi-Gui green tea. Their structures were identified by comprehensive NMR spectroscopic analyses. Absolute configurations of 1-3 were established by comparison of the CD analyses with epicatechin-3- O-gallate (ECG). Compounds 1-4 were evaluated for their protection against high glucose induced cell senescence on human umbilical vein endothelia cells (HUVECs) and showed significant protection effects ( p < 0.01) at both 1.0 and 10 µM. A discussion on the possible evolution of tea plants divergent from related food plants on the basis of phytochemical view is also provided.