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Introduction Systemic lupus erythematosus (SLE) and hypothyroidism often coexist in observational studies; however, the causal relationship between them remains controversial. Methods Complementary genetic approaches, including genetic correlation, Mendelian randomization (MR), and colocalization analysis, were conducted to assess the potential causal association between SLE and primary hypothyroidism using summary statistics from large-scale genome-wide association studies. The association between SLE and thyroid-stimulating hormone (TSH) was further analyzed to help interpret the findings. In addition, findings were verified using a validation data set, as well as through different MR methods with different model assumptions. Results The linkage disequilibrium score regression revealed a shared genetic structure between SLE and primary hypothyroidism, with the significant genetic correlation estimated to be 0.2488 (P = 6.00 × 10−4). MR analysis with the inverse variance weighted method demonstrated a bidirectional causal relationship between SLE and primary hypothyroidism. The odds ratio (OR) of SLE on primary hypothyroidism was 1.037 (95% CI, 1.013-1.061; P = 2.00 × 10−3) and that of primary hypothyroidism on SLE was 1.359 (95% CI, 1.217-1.520; P < 0.001). The OR of SLE on TSH was 1.007 (95% CI, 1.001-1.013; P = 0.032). However, TSH was not causally associated with SLE (P = 0.152). Similar results were found using different MR methods. In addition, colocalization analysis suggested that shared causal variants existed between SLE and primary hypothyroidism. The results of the validation analysis indicated a bidirectional causal relationship between SLE and primary hypothyroidism, as well as shared loci. Conclusion In summary, a bidirectional causal relationship between SLE and primary hypothyroidism was observed with complementary genetic approaches.

The Autism Mental Status exam (AMSE) has demonstrated excellent sensitivity and specificity in Western high-risk population with suspected autism spectrum disorder (ASD). This study aimed to evaluate the psychometric properties of the AMSE in a sample of high-risk Chinese children, and to determine the optimal cutoff score of the Chinese version of the AMSE in supporting ASD diagnosis. 66 young children aged from 2 to 11 years with suspected ASD were enrolled in the present study. A diagnosis of ASD or non-ASD was determined by a Best Estimate Diagnosis protocol according to the DSM-5 criteria. Receiver operating characteristic (ROC) curve analysis was conducted to assess the validity of the AMSE and search for the most effective cutoff score. The ROC curve analysis yields the area under the ROC curve of 0.98 which represents excellent diagnostic accuracy. Findings indicate the optimal cutoff score of the Chinese version of the AMSE was estimated as 6, producing the highest sensitivity of 98% and a specificity of 87%. Preliminary findings of the study suggest the AMSE has promising psychometric properties as an assessment tool for identifying ASD symptoms and supporting diagnostic decision-making in high-risk Chinese children population.

Chronic HCV infection remains a global health concern due to its involvement in hepatic and extrahepatic diseases, including B cell non-Hodgkin lymphoma (BNHL). Clinical and epidemiological evidence support a causal role for HCV in BNHL development, although mechanistic insight is lacking. We performed RNA-sequencing on peripheral B cells from patients with HCV alone, BNHL alone, and HCV-associated BNHL to identify unique and shared transcriptional profiles associated with transformation. In patients with HCV-associated BNHL, we observed the enrichment of an anergic-like gene signature and evidence of clonal expansion that was correlated with the expression of epigenetic regulatory genes. Our data support a role for viral-mediated clonal expansion of anergic-like B cells in HCV-associated BNHL development and suggest epigenetic dysregulation as a potential mechanism driving expansion. We propose epigenetic mechanisms may be involved in both HCV-associated lymphoma and regulation of B cell anergy, representing an attractive target for clinical interventions.

Objective Transcriptional profiling of immune cells is an indispensable tool in biomedical research; however, heterogenous sample types routinely used in transcriptomic studies may mask important cell type-specific transcriptional differences. Techniques to isolate desired cell types are used to overcome this limitation. We sought to evaluate the use of immunomagnetic B cell isolation on RNA quality and transcriptional output. Additionally, we aimed to develop a B cell gene signature representative of a freshly isolated B cell population to be used as a tool to verify isolation efficacy and to provide a transcriptional standard for evaluating maintenance or deviation from traditional B cell identity. Results We found RNA quality and RNA-sequencing output to be comparable between donor-matched PBMC, whole blood, and B cells following negative selection by immunomagnetic B cell isolation. Transcriptional analysis enabled the development of an 85 gene B cell signature. This signature effectively clustered isolated B cells from heterogeneous sample types in our study and naïve and memory B cells when applied to transcriptional data from a published source. Additionally, by identifying B cell signature genes whose functional role in B cells is currently unknown, our gene signature has uncovered areas for future investigation.

Purpose: Immunoassay is susceptible to interference by other substances in the serum. The main substances interfering with thyroid function testing include heterophilic antibody, biotin, thyroid hormone autoantibody, and Macro-TSH. We reported a patient with fraudulently elevated FT4 and TSH and described various common experimental methods used to explore the existence of substances interfering with thyroid function testing.Methods and Results: FT4 and TSH were significantly lower when measured on the Architect platform (FT4: 7.09 pmol/L, TSH: 58.94 µIU/ml). Polyethylene glycol precipitation showed lower FT4 and TSH, suggesting the presence of a high molecular weight interfering substance. Heterophile blocking tube study showed heterophilic antibody interfered with TSH detection. 125I-hTSH binding study and radioimmunoprecipitation assay indicated that the patient didn’t contain anti-TSH autoantibody. The new generation of Elecsys immunoassay kit indicated that biotin interfered with TSH detection. Radioimmunoprecipitation assay showed that all four kinds of thyroid hormone autoantibodies were positive. After reviewing 24 literatures, we provided the diagnostic strategy for investigation of interferences with thyroid function immunoassays.Conclusion: We reported a case with falsely elevated TSH due to the combined action of heterophilic antibody and biotin and fraudulently elevated FT4 caused by thyroid hormone autoantibody. When there is a discrepancy between thyroid function and clinical manifestation, the presence of immunoassay interference with one or more indicators needs to be considered.

Increased risk of premature cardiovascular disease (CVD) is well recognized in systemic lupus erythematosus (SLE). Aberrant type I-Interferon (IFN)-neutrophil interactions contribute to this enhanced CVD risk. In lupus animal models, the Janus kinase (JAK) inhibitor tofacitinib improves clinical features, immune dysregulation and vascular dysfunction. We conducted a randomized, double-blind, placebo-controlled clinical trial of tofacitinib in SLE subjects (ClinicalTrials.gov NCT02535689). In this study, 30 subjects are randomized to tofacitinib (5 mg twice daily) or placebo in 2:1 block. The primary outcome of this study is safety and tolerability of tofacitinib. The secondary outcomes include clinical response and mechanistic studies. The tofacitinib is found to be safe in SLE meeting study’s primary endpoint. We also show that tofacitinib improves cardiometabolic and immunologic parameters associated with the premature atherosclerosis in SLE. Tofacitinib improves high-density lipoprotein cholesterol levels (p = 0.0006, CI 95%: 4.12, 13.32) and particle number (p = 0.0008, CI 95%: 1.58, 5.33); lecithin: cholesterol acyltransferase concentration (p = 0.024, CI 95%: 1.1, −26.5), cholesterol efflux capacity (p = 0.08, CI 95%: −0.01, 0.24), improvements in arterial stiffness and endothelium-dependent vasorelaxation and decrease in type I IFN gene signature, low-density granulocytes and circulating NETs. Some of these improvements are more robust in subjects with STAT4 risk allele., Increased risk of premature cardiovascular disease in systemic lupus erythematosus (SLE) is not well understood, but in animal models, the Janus kinase inhibitor tofacitinib improves related phenotypes. Here the authors report a Phase 1 double-blind randomized trial that shows tofacitinib is safe and well tolerated in in patients with SLE.

BackgroundThere is an urgent need in clinical practice to measure the stress of parenting. The Caregiver Strain Questionnaire (CGSQ) was found to be useful to measure parenting stress, but it has not been validated among the Chinese population.AimsTo assess the reliability and construct validity of the Chinese version of CGSQ among Chinese parents.MethodsFrom 2016 to 2017, 266 parents (patient group) with a child having DSM-5–defined attention deficit hyperactivity disorder (ADHD) (n=107) or autism spectrum disorder (ASD) (n=159) and 268 parents of healthy children (control group) were recruited to the present study in Kunming, Yunnan province. All the parents were asked to fill out the Chinese version of CGSQ. We conducted exploratory factor analysis and confirmatory factor analysis (CFA) to verify construct validity of CGSQ in both patient and control groups. Cronbach’s α coefficient as an index of internal consistency was assessed for each subscale. Fourteen days later, 23 subjects filled out the scale again. Intra-class correlation coefficient was calculated to evaluate the test–retest reliability.Results(1) Cronbach’s alpha of the global scale was 0.901 for the control group and 0.952 for the patient group. The test–retest reliability for the whole scale was 0.890; (2) CFA indicated that the three-factor model had better fitting indices compared with the two-factor model in both groups. Besides, the fitting indices in the patient group were more favourable than those of the control group, with χ2/df=1.564, Goodness-of-Fit Index=0.841, Comparative Fit Index=0.954, and root mean square error of approximation=0.065 for the patient group at three-factor model; (3) The caregiver strain of ASD parents was statistically higher than that of ADHD parents, and caregiver strain of ADHD parents was higher than that of control group.ConclusionThese findings provide initial evidence to support the construct validity and reliability of CGSQ as a parenting stress measurement tool for Chinese parents, especially for parents of children with ADHD or ASD.

Changes in maternal immunity during pregnancy can result in an altered immune state, and as a natural perturbation, this provides an opportunity to understand functional interactions of the immune system in vivo. We report characterization of maternal peripheral immune phenotypes for 33 longitudinally sampled normal pregnancies, using clinical measurements of complete blood counts and major immune cell populations, as well as high parameter flow cytometry for 30 leukocyte antigens characterizing 79 cell populations, and monitoring of 1305 serum proteins using the SomaLogic platform. Cellular analyses characterized transient changes in T cell polarization and more persistent alterations in T and B cell subset frequencies and activation. Serum proteomic analysis identified a potentially novel set of 7 proteins that are predictive of gestational age: DDR1, PLAU, MRC1, ACP5, ROBO2, IGF2R, and GNS. We further show that gestational age can be predicted from the parameters obtained by complete blood count tests and clinical flow cytometry characterizing 5 major immune cell populations. Inferring gestational age from this routine clinical phenotyping data could be useful in resource-limited settings that lack obstetric ultrasound. Overall, both the cellular and proteomic analyses validate previously reported phenotypic immunological changes of pregnancy and uncover potentially new alterations and predictive markers.

Background Changes in adaptive immune cells after chemotherapy in adult acute myeloid leukemia (AML) may have implications for the success of immunotherapy. This study was designed to determine the functional capacity of the immune system in adult patients with AML who have completed chemotherapy and are potential candidates for immunotherapy. Methods We used the response to seasonal influenza vaccination as a surrogate for the robustness of the immune system in 10 AML patients in a complete remission post-chemotherapy and performed genetic, phenotypic, and functional characterization of adaptive immune cell subsets. Results Only 2 patients generated protective titers in response to vaccination, and a majority of patients had abnormal frequencies of transitional and memory B-cells. B-cell receptor sequencing showed a B-cell repertoire with little evidence of somatic hypermutation in most patients. Conversely, frequencies of T-cell populations were similar to those seen in healthy controls, and cytotoxic T-cells demonstrated antigen-specific activity after vaccination. Effector T-cells had increased PD-1 expression in AML patients least removed from chemotherapy. Conclusion Our results suggest that while some aspects of cellular immunity recover quickly, humoral immunity is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. The observed B-cell abnormalities may explain the poor response to vaccination often seen in AML patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immunity and increased PD-1 expression shortly after chemotherapy might have implications for the success of several modalities of immunotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1252-2) contains supplementary material, which is available to authorized users.

Additional file 3: Figure S2. Amplification method and Pathogen Chip assay performance assessed using positive control Viral RNAs. A) SPIA amplification vs standard (STD) method. cDNA concentration after amplification for four representative viral RNAs. Starting RNA concentration was

Multiple types of renewable distributed generation (RDG) connecting to the distribution network brings randomness to power system, which have effects on voltage stability and losses of distribution system. However, the traditional distribution planning methods cannot give consideration to both RDG capacity and installation position under various load levels. This paper proposed a fireworks optimization algorithm to obtain optimal reconfiguration of radial distribution systems with the smallest network loss (active power loss) and the maximum voltage support effect simultaneously. This algorithm can reconstruct the network and take the different RDG capacities and installation sites into overall plan. Firstly, the mathematical probability models on wind power and photovoltaic cell are established in order to consider the uncertainty of RDG. Secondly, the network switch combination added with different RDG capacities and locations are regarded as variables introduced into the fireworks algorithm. Under the consideration of various RDG operation scenarios and different load levels, the optimal sizes and positions of RDG in optimal distribution system structure can be searched by the algorithm finally. The simulation results in PE& G 69-bus system indicate the proposed method can satisfy the constraints of stable operation. In comparison of the planning only consider RDG capacities or just RDG positions, its economical superiority is well verified.

The aim of the study was to describe diagnostic stability and psychosocial outcomes of subjects with early-onset schizophrenia (EOS). All the subjects who had been hospitalized in the Department of Psychiatry of the First Affiliated Hospital of Kunming Medical University between January 2011 and July 2015 with the diagnosis of International Classification of Diseases (ICD)-10 defined schizophrenia, and discharged from the hospital for more than 12 months were enrolled to the study. The Mini International Neuropsychiatric Interview was applied for life-time ICD -10 diagnoses, and Personal and Social Performance (PSP) for global function evaluation. Altogether 249 patients were targeted for follow-up, in which 101 were followed up and the dropout rate was 59.4%. After average 37.2 ± 16.2 months, 92 patients (including 1 death) were still met the ICD-10 diagnosis of schizophrenia (F20). In terms of global functioning, 48.5% of patients had good outcome, 43.6% had moderate outcome, and 7.9% had poor outcome. A relatively high diagnostic stability of ICD-10 defined schizophrenia was obtained in the current study .Moreover, our results draw a much more optimistic picture of the outcome for EOS than what has previously been reported from Western counties.

Additional file 6: Figure S3. tSNE projection of live and fixed PBMCs from donor DTM-X (a) and donor (b). Cells were grouped using graph-based method. Classification of PBMCs was inferred from the annotation of cluster-specific genes, and based on expression of some well-known markers of immune cell types. Although fixation lead to changes of the relative distances of the clusters due to the loss of genes detected, it did not impact the resolution of the low abundant populations (B, NK, DC) in each sample. Subpopulations were detected from fixed PBMCs at a similar proportion to those of live PBMCs (Table 1).

Additional file 3. Supplemental figures.

Scientific Reports 6: Article number: 23002; published online: 14 March 2016; updated: 04 May 2016 This Article contains errors in the Author Contributions Statement, “J.S.T. headed and interpreted the data analysis, revised the manuscript.” should read: “J.S.T helped design data analysis strategiesand analysis result interpretation, and contributed to manuscript revision.

Corticosteroids have been used for decades to modulate inflammation therapeutically, yet there is a paucity of data on their effects in humans. We examined the changes in cellular and molecular immune system parameters, or “immunome”, in healthy humans after systemic corticosteroid administration. We used multiplexed techniques to query the immunome in 20 volunteers at baseline, and after intravenous hydrocortisone (HC) administered at moderate (250 mg) and low (50 mg) doses, to provide insight into how corticosteroids exert their effects. We performed comprehensive phenotyping of 120 lymphocyte subsets by high dimensional flow cytometry, and observed a decline in circulating specific B and T cell subsets, which reached their nadir 4–8 hours after administration of HC. However, B and T cells rebounded above baseline 24 hours after HC infusion, while NK cell numbers remained stable. Whole transcriptome profiling revealed down regulation of NF-κB signaling, apoptosis, and cell death signaling transcripts that preceded lymphocyte population changes, with activation of NK cell and glucocorticoid receptor signaling transcripts. Our study is the first to systematically characterize the effects of corticosteroids on the human immunome, and we demonstrate that HC exerts differential effects on B and T lymphocytes and natural killer cells in humans.

Twenty natural dyes, extracted from natural materials such as flowers, leaves, fruits, traditional Chinese medicines, and beverages, were used as sensitizers to fabricate dye-sensitized solar cells (DSCs). The photoelectrochemical performance of the DSCs based on these dyes showed that the open circuit voltages (Voc) varied from 0.337 to 0.689 V, and the short circuit photocurrent densities (Jsc) ranged from 0.14 to 2.69 mA cm −2 . Specifically, a high Voc of 0.686 V was obtained from the dye extracted from mangosteen pericarp sensitizer. The photo-to-electric conversion efficiency of the DSC sensitized by the ethanol extract of mangosteen pericarp without purification reached 1.17%. Moreover, various components of the ethanol extract were extracted using different organic solvents. The photoelectrochemical performance of these extracts demonstrated that rutin was the most effectual component of the sensitizer for DSC. © 2011 Elsevier B.V. All rights reserved.

A new class of organic dyes based on acenaphthopyrazine derivatives, containing pyrazine group as the electron acceptor and o-dicarboxyl acids as the anchoring groups were designed and synthesized ...

Hibernating mammals survive hypothermia (10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

The power system islanding problem aims to divide the power system into several different islands after serious disturbances occur. The objective of this problem is to minimize the total generation-load imbalance of all islands while placing the coherent generators in the same island and maintaining the connectivity of each island. Two main challenges of solving this problem are the large scale of the power system and the requirement of a short computation time. In this study, we propose a tailored tabu search algorithm to solve this problem, which employs a novel initial solution generation procedure and a neighborhood operation based on the movement of the boundary nodes. The numerical experiments on 15 test instances show that this algorithm can deal with the power systems with up to 3120 nodes within 0.7 s. Then, the comparisons with some existing islanding methods based on the IEEE 39-bus system and the IEEE 118-bus system prove the validity and accuracy of our method. Finally, time-domain simulations based on three power systems demonstrate the importance of our tabu search algorithm in minimizing the impacts of the disastrous disturbances. The computational results imply that our tabu search algorithm is very effective and efficient and satisfies the requirements of islanding power systems of various sizes.

SummaryA major goal of systems biology is the development of models that accurately predict responses to perturbation. Constructing such models requires the collection of dense measurements of system states, yet transformation of data into predictive constructs remains a challenge. To begin to model human immunity, we analyzed immune parameters in depth both at baseline and in response to influenza vaccination. Peripheral blood mononuclear cell transcriptomes, serum titers, cell subpopulation frequencies, and B cell responses were assessed in 63 individuals before and after vaccination and were used to develop a systematic framework to dissect inter- and intra-individual variation and build predictive models of postvaccination antibody responses. Strikingly, independent of age and pre-existing antibody titers, accurate models could be constructed using pre-perturbation cell populations alone, which were validated using independent baseline time points. Most of the parameters contributing to prediction delineated temporally stable baseline differences across individuals, raising the prospect of immune monitoring before intervention.

Meeting abstracts High-throughput sequencing technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. They provided in-depth of new information that can reframe our understanding of human cancer

The immunotherapy of cancer is arguably the most promising therapy under development, and vaccination against cancer antigens is a promising strategy to stimulate adaptive immune responses against malignant clones. However, the ability of patients treated with chemotherapy to respond appropriately to immune challenge may be suboptimal. This study was designed to determine the functional capacity of the immune system in adult acute myeloid leukemia (AML) patients who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to influenza vaccination as a surrogate for the health of the immune system in AML patients in a complete remission (CR) post-chemotherapy. Ten adult AML patients in CR after completion of intensive chemotherapy were recruited to the clinical protocol J1293. They were on average 37 weeks post-treatment (range 4-148) when receiving the 2012-2013 inactivated seasonal influenza vaccine. Peripheral blood samples were collected at baseline and 30 days post-vaccination. Ten age and sex matched healthy donors (HD) served as baseline controls. Serological response to vaccination was assessed via microneutralization assays; multi-parameter flow cytometry was used to characterize lymphocyte subsets. ELISPOT assays were used to evaluate lymphocyte function, microarrays were used to assess gene expression, and deep sequencing of the B-cell receptor heavy chain (IGH) was performed to determine expansion and clonality of B-cells. Wilcoxon rank-sum tests were used to assess statistical significance. Only 2/10 (20%) patients seroconverted (AML responders, or AML-R) with a four-fold increase or greater in influenza-specific antibody. One responder was 148 weeks post-treatment; the other had acute promyelocytic leukemia (APL) and was 4 weeks post-treatment. Deep immunophenotyping revealed no striking differences in T-cell compartments between AML at baseline and HD, indicating rapid T-cell recovery after chemotherapy. In contrast, we observed a highly atypical B-cell profile. AML non-responders (AML-NR) at day 0 versus HD had significantly reduced frequencies of mature IgA+ (4.5% vs. 11%) and IgG+ (2.4% vs. 5.9%) B-cells (as a percentage of CD19+CD20+). Further dissection exposed markedly higher frequencies of CD10+CD27- transitional B-cells (36% vs. 16%, p Interestingly, functional T-cell assays revealed that of 5 evaluable patients, including the 2 AML-R and 3 AML-NR, 5/5 (100%) patients had an increase in influenza-specific cytokine production (1.24 - 4.40x higher on day 30 over baseline), suggesting a functional T-cell response even with deficient influenza-specific antibody production. Supervised clustering of microarray data identified many upregulated genes in AML-NR related to apoptosis, BCR, IL2, IL-4, IL-8, and IL-12 signaling pathways, indicative of developing B-cells. IGH sequencing demonstrated AML-NR had greater variability in CDR3 length than seen in HD, consistent with an antigen inexperienced B-cell repertoire. These data suggest that while some aspects of cellular immunity recover comparatively quickly, the humoral immune system is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. Abnormal frequencies of transitional and memory B-cells may explain the poor response to vaccination often seen in patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immune capacity observed here might have implications for the success of immunotherapies based on vaccination. Figure 1. Figure 1. Disclosures Noonan: Celgene: Speakers Bureau. Borrello:Celgene: Research Funding.

A newly developed photoelectrochemistry analytical methodology was applied to selective detection of the immunoreaction between anti human IgG and human IgG. The method is based on the principle of the dye-sensitized solar cell, using an NHS-ester derivative of ruthenium polypyridine as the signal indicator and nanoparticle TiO 2 as electrode material. After performing the affinity reaction, photoelectrochemical measurement showed that anodic photocurrent was turned on and off repeatedly by control of incidental visible-light, due to light absorption of the ruthenium compound immobilized on the electrode. Owing to its separate source for excitation (light) and detection (current) and the use of electronic detection, the photoelectrochemistry system is expected as a sensitive and low cost method for the detection of biological affinity interaction.

Dye-sensitized solar cell (DSC) is a new class of green photovoltaic cell based on photosynthesis principle in nature. We fabricated the DSCs using ten natural dyes as sensitizers, which extracted from the materials existing in nature and our life, such as flowers, leaves, fruits, traditional Chinese medicines, and beverages. The photoelectrochemical performances of the DSCs based on the dyes showed that the open circuit voltages (Voc) varied from 0.49–0.69 V and the short circuit photocurrent densities (Jsc) were in the range of 0.74–2.69 mA/cm2. It is expected that the energy conversion efficiency can be further improved by introducing functional group and optimizing structure of natural dye.