The bidirectional temporal relationship between parenting stress and child maltreatment: A cross-lagged study based on intervention and cohort data.

BACKGROUND: Parenting stress has long been proposed as a major risk factor for child maltreatment. However, there is a lack of evidence from existing studies on the temporal sequence to establish a causal relationship. This study aims to examine bidirectional temporal relationships between parenting stress and child maltreatment. METHODS: Longitudinal data from two different sources were analysed: a pre-post study of an online parenting programme conducted across six countries - the ePLH Evaluation Study, and a prospective cohort study in the United States - LONGSCAN. Cross-lagged panel model on parenting stress and child maltreatment was used in each dataset. RESULTS: Based on repeatedly measured data of 484 caregivers in the ePLH study across five time points (every two weeks), we found that parenting stress at an earlier time point predicted later child maltreatment (IRR = 1.14, 95 % CI: 1.10,1.18). In addition, the occurrence of child maltreatment was associated with higher subsequent short-term parenting stress (IRR = 1.04, 95 % CI: 1.01,1.08) and thus could form a vicious circle. In the LONGSCAN analysis with 772 caregivers who were followed up from child age of 6 to child age of 16, we also found parenting stress at an earlier time point predicted later child maltreatment (ß = 0.11, 95 % CI: 0.01,0.20), but did not observe an association between child maltreatment and subsequent long-term parenting stress. LIMITATIONS: Potential information bias on the measurements. CONCLUSIONS: This study provides evidence for a bidirectional temporal relationship between parenting stress and child maltreatment, which should be considered in parenting intervention programmes.

NLRC4 methylation and its response to intravenous immunoglobulin therapy in Kawasaki disease: a case control study.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis accompanied by many systemic physiological and biochemical changes. Elucidating its molecular mechanisms is crucial for diagnosing and developing effective treatments. NLR Family CARD Domain Containing 4 (NLRC4) encodes the key components of inflammasomes that function as pattern recognition receptors. The purpose of this study was to investigate the potential of NLRC4 methylation as a biomarker for KD. METHODS: In this study, pyrosequencing was utilized to analyze NLRC4 promoter methylation in blood samples from 44 children with initial complete KD and 51 matched healthy controls. Methylation at five CpG sites within the NLRC4 promoter region was evaluated. RESULTS: Compared to controls, NLRC4 methylation significantly decreased in KD patients (CpG1: p = 2.93E-06; CpG2: p = 2.35E-05; CpG3: p = 6.46E-06; CpG4: p = 2.47E-06; CpG5: p = 1.26E-05; average methylation: p = 5.42E-06). These changes were significantly reversed after intravenous immunoglobulin (IVIG) treatment. ROC curve analysis demonstrated remarkable diagnostic capability of mean NLRC4 gene methylation for KD (areas under ROC curve = 0.844, sensitivity = 0.75, p = 9.61E-06, 95% confidence intervals were 0.762-0.926 for mean NLRC4 methylation). In addition, NLRC4 promoter methylation was shown to be significantly negatively correlated with the levels of central granulocyte percentage, age, mean haemoglobin quantity and mean erythrocyte volume. Besides, NLRC4 promoter methylation was positively correlated with lymphocyte percentage, lymphocyte absolute value. CONCLUSIONS: Our work revealed the role of peripheral NLRC4 hypomethylation in KD pathogenesis and IVIG treatment response, could potentially serve as a treatment monitoring biomarker, although its precise functions remain to be elucidated.

Necrotizing apoptosis-related genes prognosis and treatment effect analysis of osteosarcoma in children.

BACKGROUND: Immune cell homeostasis plays a crucial role in cancer research and therapeutic response. While chemotherapy and immunotherapy hold promise in treating osteosarcoma (OS), identifying patients who are likely to respond would significantly improve clinical practices. Necroptosis, a fundamental mechanism mediating chemotherapy and immunotherapy efficacy, offers valuable insights. In this context, subtypes based on necroptosis-related genes have been established to predict the response of OS patients to immunotherapy and chemotherapy. METHODS: We conducted a high-throughput screening test to identify necroptosis-associated genes that regulate the development of osteosarcoma. Subsequently, the ConsensusClusterPlus package was employed to classify OS patients into subtypes, enabling comparisons of prognosis and clinical information between these subtypes. Patients from the TARGET-OS and GSE21257 datasets were stratified into high-risk and low-risk groups, and their prognoses were compared. Additionally, we assessed the accuracy of the Risk Scoring Model in predicting prognosis, identified independent prognostic factors and explored potential chemotherapeutic agents and immunotherapy drugs. RESULTS: Through the intersection of expression profiles from the TARGET-OS and GSE21257 datasets, we have identified a total of 92 genes associated with necroptosis. Based on differences in the expression of these genes, patients were divided into three subtypes, and we investigated the differences in tumor-infiltrating immune cells, immune-related pathways, and prognosis among these subtypes. Our nomogram effectively differentiated subtypes with distinct responses to chemotherapy and immunotherapy. The established signature demonstrated superior prediction ability compared with single clinical indicators. CONCLUSIONS: This pioneering study unveils the prognostic role of necroptosis-related genes in OS patients, providing a promising alternative for prognostic prediction in clinical disease management. Moreover, our findings highlight the significance of immune cell homeostasis in cancer research and therapeutic response, underscoring its relevance in advancing current treatment strategies.

Do Resources Help or Hurt? The Critical Roles of Me, Others, and Organization in Mitigating Workplace Demands.

Today's job demands-resources (JD-R) literature emphasizes the interactions between job demands and resources. However, the dynamism of JD-R processes in which individuals manage to counteract the detrimental effects of job demands on employees by flexibly adjusting resources calls this relationship into question. Hence, in this study, we depict JD-R dynamics in the workplace by developing a cross-level moderated-mediation model that unveils the different roles played by job and personal resources when they are woven together with job demands. We examine how and when the relationships among three types of resources-organizationally assigned high-performance work systems (HPWSs), psychological capital (PsyCap), and social support climate (SSC)-help employees deal with excessive workload and unhealthy emotional demands. Using a two-wave research design, we measured and tested the causal relationships of the proposed variables. We found that (1) HPWSs had a negative effect on workload, (2) workload provoked employee emotional demands, (3) the interaction between HPWSs and PsyCap increased workload, and (4) SSC mitigated the positive effect of workload on employee emotional demands. This study contributes to the JD-R theory and literature by unlocking the potential roles of various resources. Theoretical and practical implications, limitations, and future research avenues are also presented.

The Identification of Viral Pathogens in a Physostegia virginiana Plant Using High-Throughput RNA Sequencing.

Physostegia virginiana is an important ornamental and cut-flower plant in China. Its commonly used method of clonal propagation leads to virus accumulation in this plant. However, which viruses can infect the Physostegia virginiana plant remains to be illuminated. In this work, five viral pathogens in a Physostegia virginiana plant with virus-like symptoms of yellow, shriveled, and curled leaves were identified using RNA-seq, bioinformatics, and molecular biological techniques. These techniques allowed us to identify five viruses comprising one known alfalfa mosaic virus (AMV) and four novel viruses. The novel viruses include a virus belonging to the genus Fabavirus, temporarily named Physostegia virginiana crinkle-associated virus 1 (PVCaV1); two viruses belonging to the genus Caulimovirus, temporarily named Physostegia virginiana caulimovirus 1 and 2 (PVCV1 and PVCV2); and a virus belonging to the genus Fijivirus, temporarily named Physostegia virginiana fijivirus (PVFV). The genome sequences of PVCaV1, PVCV1, and PVCV2, and the partial genome sequence of PVFV were identified. Genome organizations and genetic evolutionary relationships of all four novel viruses were analyzed. PVCaV1 has a relatively close evolutionary relationship with five analyzed fabiviruses. PVCV1 and PVCV2 have separately a closest evolutionary relationship with lamium leaf distortion-associated virus (LLDAV) and figwort mosaic virus (FMV), and PVFV has a close evolutionary relationship with the five analyzed fijiviruses. Additionally, PVCaV1 can infect Nicotiana benthamiana plants via friction inoculation. The findings enrich our understanding of Physostegia virginiana viruses and contribute to the prevention and control of Physostegia virginiana viral diseases.

Overexpression of maize GOLDEN2 in rice and maize calli improves regeneration by activating chloroplast development.

Golden2 (G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene (rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

Efficacy and safety of etanercept biosimilar rhTNFR-Fc in Chinese patients with juvenile idiopathic arthritis: An open-label multicenter observational study.

Background: Etanercept biosimilar recombinant human TNF-α receptor II: IgG Fc fusion protein (rhTNFR-Fc) has showed its efficacy and safety in Chinese patients with rheumatoid arthritis. However, data on rhTNFR-Fc's application in juvenile idiopathic arthritis (JIA) is limited. Methods: A prospective, observational, multicenter study was performed at 6 institutes in China from July 2020 to December 2021. In a 24-week follow-up, patients with JIA including polyarticular JIA and enthesitis related arthritis received rhTNFR-Fc plus methotrexate (MTX) treatment. The primary outcome parameters were improvements of cJADAS-10 (clinical Juvenile Arthritis Disease Activity Score), and the secondary outcome parameter was an inactive disease. Results: 60 patients completed at least 12-week follow-up, and 57 completed 24-week follow-up. They had high C reactive protein values (11.6 mg/L) and cJADAS-10 (14.6) at baseline. Thirteen patients had morning stiffness. 33 patients showed synovial thickening, and 34 showed bone marrow edemas on MRI. Ultrasonography demonstrated significant joint effusions in 43 patients. The cJADAS-10 sharply decreased from 14.66 at the baseline to 2.4 at 24 weeks of rhTNFR-Fc therapy, respectively (P < 0.01). About half of patients achieved inactive disease at 24 weeks of therapy. Compared with the baseline, the number of patients with morning stiffness, joint effusions, bone marrow edema and synovial thickening on MRI significantly decreased at 24 weeks. Adverse events were consistent with known side effects of biologic agents. Conclusions: The present study indicated that the combination of rhTNFR-Fc and MTX significantly improve symptoms and disease activity of children with JIA. This study suggests etanercept biosimilar rhTNFR-Fc as an effective and safe therapy for children with JIA.

Blocking Rice Shoot Gravitropism by Altering One Amino Acid in LAZY1.

Tiller angle is an important trait that determines plant architecture and yield in cereal crops. Tiller angle is partially controlled during gravistimulation by the dynamic re-allocation of LAZY1 (LA1) protein between the nucleus and plasma membrane, but the underlying mechanism remains unclear. In this study, we identified and characterized a new allele of LA1 based on analysis of a rice (Oryza sativa L.) spreading-tiller mutant la1G74V, which harbors a non-synonymous mutation in the predicted transmembrane (TM) domain-encoding region of this gene. The mutation causes complete loss of shoot gravitropism, leading to prostrate growth of plants. Our results showed that LA1 localizes not only to the nucleus and plasma membrane but also to the endoplasmic reticulum. Removal of the TM domain in LA1 showed spreading-tiller phenotype of plants similar to la1G74V but did not affect the plasma membrane localization; thus, making it distinct from its ortholog ZmLA1 in Zea mays. Therefore, we propose that the TM domain is indispensable for the biological function of LA1, but this domain does not determine the localization of the protein to the plasma membrane. Our study provides new insights into the LA1-mediated regulation of shoot gravitropism.

Metabolomics of bronchoalveolar lavage in children with persistent wheezing.

BACKGROUND: Recent studies have demonstrated the important role of metabolomics in the pathogenesis of asthma. However, the role of lung metabolomics in childhood persistent wheezing (PW) or wheezing recurrence remains poorly understood. METHODS: In this prospective observational study, we performed a liquid chromatography/mass spectrometry-based metabolomic survey on bronchoalveolar lavage samples collected from 30 children with PW and 30 age-matched infants (control group). A 2-year follow-up study on these PW children was conducted. RESULTS: Children with PW showed a distinct characterization of respiratory metabolome compared with control group. Children with PW had higher abundances of choline, oleamide, nepetalactam, butyrylcarnitine, L-palmitoylcarnitine, palmitoylethanolamide, and various phosphatidylcholines. The glycerophospholipid metabolism pathway was the most relevant pathway involving in PW pathophysiologic process. Additionally, different gender, prematurity, and systemic corticoids use demonstrated a greater impact in airway metabolite compositions. Furthermore, for PW children with recurrence during the follow-up period, children who were born prematurely had an increased abundance of butyrylcarnitine relative to those who were carried to term. CONCLUSIONS: This study suggests that the alterations of lung metabolites could be associated with the development of wheezing, and this early alteration could also be correlated with wheezing recurrence later in life.

Follow-up study of airway microbiota in children with persistent wheezing.

BACKGROUND: Increasing evidence revealed that airway microbial dysbiosis was associated with increased risk of asthma, or persistent wheezing (PW). However, the role of lung microbiota in PW or wheezing recurrence remains poorly understood. METHODS: In this prospective observational study, we performed a longitudinal 16S rRNA-based microbiome survey on bronchoalveolar lavage (BAL) samples collected from 35 infants with PW and 28 age-matched infants (control group). A 2-year follow-up study on these PW patients was conducted. The compositions of lower airway microbiota were analyzed at the phylum and genus levels. RESULTS: Our study showed a clear difference in lower airway microbiota between PW children and the control group. Children with PW had a higher abundance of Elizabethkingia and Rothia, and lower abundance of Fusobacterium compared with the control group. At the end of the 2-year follow-up, 20 children with PW (57.1%) experienced at least one episode of wheezing, and 15 (42.9%) did not suffer from wheezing episodes. Furthermore, PW children with recurrence also had increased abundances of Elizabethkingia and Rothia relative to those who had no recurrence. Additionally, wheezing history, different gender, and caesarean section demonstrated a greater impact in airway microbiota compositions. CONCLUSION: This study suggests that the alterations of lower airway microbiota could be strongly associated with the development of wheezing, and early airway microbial changes could also be associated with wheezing recurrence later in life.

A lipid transfer protein variant with a mutant eight-cysteine motif causes photoperiod- and thermo-sensitive dwarfism in rice.

Plant height is an important trait for architecture patterning and crop yield improvement. Although the pathways involving gibberellins and brassinosteroids have been well studied, there are still many gaps in our knowledge of the networks that control plant height. In this study, we determined that a dominant photoperiod- and thermo-sensitive dwarf mutant is caused by the active role of a mutated gene Photoperiod-thermo-sensitive dwarfism 1 (Ptd1), the wild-type of which encodes a non-specific lipid transfer protein (nsLTP). Ptd1 plants showed severe dwarfism under long-day and low-temperature conditions, but grew almost normal under short-day and high-temperature conditions. These phenotypic variations were associated with Ptd1 mRNA levels and accumulation of the corresponding protein. Furthermore, we found that the growth inhibition in Ptd1 may result from the particular protein conformation of Ptd1 due to loss of two disulfide bonds in the eight-cysteine motif (8-CM) that is conserved among nsLTPs. These results contribute to our understanding of the novel function of disulfide bonds in the 8-CM, and provide a potential new strategy for regulation of cell development and plant height by modifying the amino acid residues involved in protein conformation patterning.

Correction to: Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula.

[This corrects the article DOI: 10.1186/s13007-019-0404-1.].

Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula.

BACKGROUND: Tissue culture transformation of plants has an element of art to it, with protocols passed on between labs but often not directly compared. As Medicago truncatula has become popular as a model system for legumes, rapid transformation is critical, and many protocols exist, with varying results. RESULTS: The M. truncatula ecotypes, R108 and A17, were utilized to compare the effect of a modification to a previously used protocol based on shoot explants on the percentage of transformed plants produced from calli. This percentage was then compared to that of two additional transformation protocols based on root explants in the R108 ecotype. Variations in embryonic tissue sources, media components, time for transformation, and vectors were analyzed. CONCLUSIONS: While no A17 transgenic plants were obtained, transgenic plantlets from the R108 ecotype were produced in as little as 4 months with a comparison of the two widely studied ecotypes under a single set of conditions. While the protocols tested gave similar results in percentage of transformed plants produced, considerations of labor and time to transgenics that vary between the root explant protocols tested were discovered. These considerations may influence which protocol to choose for introducing a single transgene versus creating lines with multiple mutations utilizing a CRISPR/Cas9 construct.

The Intronic cis Element SE1 Recruits trans-Acting Repressor Complexes to Repress the Expression of ELONGATED UPPERMOST INTERNODE1 in Rice.

Plant height has a major effect on grain yield in crops such as rice (Oryza sativa), and the hormone gibberellic acid (GA) regulates many developmental processes that feed into plant height. Rice ELONGATED UPPERMOST INTERNODE1 (Eui1) encodes a GA-deactivating enzyme governing elongation of the uppermost internode. The expression of Eui1 is finely tuned, thereby maintaining homeostasis of endogenous bioactive GA and producing plants of normal plant height. Here, we identified a dominant dwarf mutant, dEui1, caused by the deletion of an RY motif-containing cis-silencing element (SE1) in the intron of Eui1. Detailed genetic and molecular analysis of SE1 revealed that this intronic cis element recruits at least one trans-acting repressor complex, containing the B3 repressors OsVAL2 and OsGD1, the SAP18 co-repressor, and the histone deacetylase OsHDA710, to negatively regulate the expression of Eui1. This complex generates closed chromatin at Eui1, suppressing Eui1 expression and modulating GA homeostasis. Loss of SE1 or dysfunction of the complex components impairs histone deacetylation and H3K27me3 methylation of Eui1 chromatin, thereby increasing Eui1 transcription and decreasing bioactive GA, producing dwarfism in rice. Together, our results reveal a novel silencing mechanism in which the intronic cis element SE1 negatively regulates Eui1 expression via repressor complexes that modulate histone deacetylation and/or methylation.

Elevated Interleukin 1ß and Interleukin 6 Levels in the Serum of Children With Hyperuricemia.

PURPOSES: The aim of this study was to investigate the serum levels and clinical significance of interleukin 1ß (IL-1ß) and IL-6 in children with hyperuricemia (HUA). METHODS: We included 71 children with HUA and 71 children with no HUA as control subjects. Children with HUA were divided into groups I and II according to whether they had a history of acute gout-like attacks (including sudden monoarthritis of rapid onset with intense pain and swelling). Group I was examined twice (A, in the acute phase; B, in the remission phase). Serum IL-1ß and IL-6 levels were measured by enzyme-linked immunosorbent assay. RESULTS: Serum IL-1ß and IL-6 levels were increased in children with HUA and were overall statistically different from the control group (P < 0.05, respectively). Serum IL-1ß and IL-6 were significantly higher in group IA in comparison with group IB, group II, and control subjects (P < 0.05, respectively), as well as in groups IB and II compared with control subjects (P < 0.05, respectively). In group IB, the serum IL-1ß and IL-6 concentrations were higher than those in group II, but there were no statistical differences (P > 0.05). In addition, in children with HUA, serum IL-1ß and IL-6 levels were positively associated with white blood cell count, neutrophil count, monocyte count, uric acid levels, erythrocyte sedimentation rate, C-reactive protein, blood urea nitrogen, and serum creatinine levels (all P < 0.05), but were not associated with triglycerides, total cholesterol, low-density lipoprotein cholesterol, or high-density lipoprotein cholesterol levels (all P > 0.05). CONCLUSION: IL-1ß and IL-6 levels are increased in children with hyperuricemia, even if they have not had acute gout. Further studies are necessary to fully characterize the significance of IL-1ß and IL-6 found in HUA children, and whether they could be correlated with long-term prognosis.

Elevated urinary monocyte chemoattractant protein-1 levels in children with Henoch-Schonlein purpura nephritis.

BACKGROUND: Chemokine monocyte chemoattractant protein-1 (MCP-1) has been proved as a potential urinary biomarker in nephropathies. The aim of this study was to investigate the urinary monocyte chemoattractant protein-1 (MCP-1) levels and clinical significance in Henoch-Schonlein purpura (HSP) children with and without nephritis and determine the association of MCP-1 with proteinuria. METHODS: A total of 261 HSP children-with or without nephritis-and 84 healthy control children were enrolled in this study. Of these, 126 HSP nephritis (HSPN) children were subdivided into three groups according to total urine protein in 24 h (TUP): Group A, mild proteinuria group with TUP <25 mg/kg; Group B, moderate proteinuria group with TUP ≥25 mg/kg and <50 mg/kg; Group C, severe proteinuria group with TUP ≥50 mg/kg. Urinary MCP-1 levels were determined by ELISA. Levels of serum creatinine (Cr), blood urea nitrogen (BUN), urinary α1-micro globulin (α1-MG), micro-albumin (mAlb), immunoglobulin G (IgG), transferrin (TRF) and TUP were performed to determine their associations with MCP-1. RESULTS: Urinary MCP-1 was significantly higher in HSPN group in comparison with HSP group and controls (P < 0.05), but no significant difference was found between the HSP group and the healthy group (P > 0.05). The levels of urinary MCP-1 increased in parallel to the enhancement of total urine protein in 24 h in HSPN patients. There were statistically significant differences among these three groups of HSPN children (p < 0.05). Urinary MCP-1 correlated positively with urinary α1-MG, mAlb, IgG, TRF and TUP in HSPN, whereas no correlation was observed with serum Cr and BUN. CONCLUSIONS: MCP-1 was elevated in children with HSPN and correlated with proteinuria. Urinary MCP-1 could be used as a suitable, non-invasive biomarker to provide valuable information not only for the diagnosis of HSPN, but also for evaluation of severity of renal damage.

Urinary Macrophage Migration Inhibitory Factor as a Noninvasive Biomarker in Pediatric Henoch-Schönlein Purpura Nephritis.

PURPOSES: The aims of this study were to investigate urinary macrophage migration inhibitory factor (MIF) levels and their clinical significance in Henoch-Schönlein purpura (HSP) children with or without nephritis (N) and to assess the influence of steroid treatment on the urine MIF levels of HSPN patients. METHODS: Group I comprised 35 children with HSPN who were examined twice (A before treatment and B after steroid treatment). Group II comprised 41 children with HSP. The control group included 32 healthy children. Urinary MIF levels were measured via enzyme linked immunosorbent assay. The levels of serum creatinine, blood urea nitrogen, urinary microalbumin (mAlb), and 24-hour proteinuria were performed to determine their associations with MIF levels. RESULTS: Urinary MIF levels were significantly higher in group I compared with group II and the control group (P < 0.01); however, no significant difference was found between group II and the control group (P > 0.05). Upon examination, albeit urinary MIF concentration was significantly lower in group IB compared with group IA (P < 0.05), these concentrations were statistically higher than that of group II (P < 0.05). In addition, in the HSPN patients, the urinary MIF was positively associated with urinary microalbumin and 24-hour proteinuria but no association with serum creatinine and blood urea nitrogen. CONCLUSIONS: Elevated urinary MIF levels were found to be correlated with proteinuria in pediatric HSPN. An obvious decrease in urinary MIF concentrations among the children with HSPN was associated with steroid treatment. Urinary MIF can be used as a noninvasive biomarker in pediatric HSPN.

Development of "Purple Endosperm Rice" by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System.

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.