Expression inheritance and constraints on cis- and trans-regulatory mutations underlying lotus color variation.

Both cis- and trans-regulatory mutations drive changes in gene expression that underpin plant phenotypic evolution. However, how and why these two major types of regulatory mutations arise in different genes and how gene expression is inherited and associated with these regulatory changes are unclear. Here, by studying allele-specific expression in F1 hybrids of pink-flowered sacred lotus (Nelumbo nucifera) and yellow-flowered American lotus (N. lutea), we reveal the relative contributions of cis- and trans-regulatory changes to interspecific expression rewiring underlying petal color change and how the expression is inherited in hybrids. Although cis-only variants influenced slightly more genes, trans-only variants had a stronger impact on expression differences between species. In F1 hybrids, genes under cis-only and trans-only regulatory effects showed a propensity toward additive and dominant inheritance, respectively, whereas transgressive inheritance was observed in genes carrying both cis- and trans-variants acting in opposite directions. By investigating anthocyanin and carotenoid coexpression networks in petals, we found that the same category of regulatory mutations, particularly trans-variants, tend to rewire hub genes in coexpression modules underpinning flower color differentiation between species; we identified 45 known genes with cis- and trans-regulatory variants significantly correlated with flower coloration, such as ANTHOCYANIN 5-AROMATIC ACYLTRANSFERASE (ACT), GLUTATHIONE S-TRANSFERASE F11 (GSTF11), and LYCOPENE Ε-CYCLASE (LCYE). Notably, the relative abundance of genes in different categories of regulatory divergence was associated with the inferred magnitude of constraints like expression level and breadth. Overall, our study suggests distinct selective constraints and modes of gene expression inheritance among different regulatory mutations underlying lotus petal color divergence.

Evaluation of a Novel Formaldehyde-Free Fixation Solution for the Fixation of Mouse Organs.

Objective: A new aldehyde-free fixative has been developed and its effect has been compared to traditional formaldehyde fixative in terms of the fixation effect and HE staining of the heart, liver, lung, and kidney. The air in the experimental area was examined to evaluate its impact on the environment and human health. Methods: The organs from mice of groups 1-6 were taken respectively (thickness of liver and lung was 3 mm). After the heart and kidney capsule were removed, the organs were longitudinally cut along their maximum surface, and half was taken. Thereafter, the tissue fixation effect was observed by Hematein and Eosin (H＆E) staining and the total protein content of tissue was examined by the ultramicro spectrophotometer. Additionally, the volatility ratio of the new fixative and the traditional formaldehyde is compared. Result: The results showed that there was no significant difference between the fixation effect of the new aldehyde-free fixation and the traditional formaldehyde fixative on mouse organs and the air quality in the experimental area was found to be significantly better when the new aldehyde-free fixative is used than when the traditional formaldehyde fixative is used. Conclusion: Traditional formaldehyde fixative in HE staining can be replaced by the new environment-friendly formaldehyde-free fixative, however further special staining of fixed tissue and immunohistochemical studies are needed.

Autophagy of hepatic stellate cell induced by Clonorchis sinensis.

BACKGROUND: Clonorchis sinensis was a food-borne zoonotic parasite in the worldwide and also an important risk factor of hepatic fibrosis. Excretory/secretion products of C. sinensis (CsESPs) are involved in parasite-host interactions and contribute to the development of hepatic damage. The aim of the present study was to investigate whether CsESPs and CsTP (adult protein) could induce autophagy of hepatic stellate cells (HSCs) and further activate HSCs so as to participate in the pathogenesis of hepatic fibrosis. METHODS AND RESULTS: The human hepatic stellate cell line LX-2 was stimulated by CsESPs and CsTP. CsESPs showed the effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay while CsTP failed. Autophagosomes and autolysosomes were observed after the transmission mRFP-EGFP-LC3 plasmid into the LX-2 cells. CsESPs had more powerful to induce the accumulation of autophagosomes and autolysosomes to enhance autophagic flux compared with CsTP. Western-blotting analysis confirmed that the ratio of LC3-II/I in LX-2 cells was up-regulated after CsESPs treatment for 6 h, which further proved that CsESPs could induce autophagy in LX-2 cells. Meanwhile, q-PCR results showed that the mRNA levels of collagen I, collagen III and α-SMA decreased in LX-2 cells after treatment with autophagy inhibitor chloroquine, whereas they increased when combination with CsESPs. CONCLUSIONS: These results suggested that CsESPs-induced autophagy might be involved in the activation of HSCs, and consequently participate in the pathogenesis of hepatic fibrosis caused by C. sinensis infection.

Biased allelic expression in tissues of F1 hybrids between tropical and temperate lotus (Nelumbo nuicfera).

KEY MESSAGE: The genome-wide allele-specific expression in F1 hybrids from the cross of tropical and temperate lotus unveils how cis-regulatory divergences affect genes in key pathways related to ecotypic divergence. Genetic variation, particularly cis-regulatory variation, plays a crucial role in phenotypic variation and adaptive evolution in plants. Temperate and tropical lotus, the two ecotypes of Nelumbo nucifera, show distinction in the degree of rhizome enlargement, which is associated with winter dormancy. To understand the roles of genome-wide cis-regulatory divergences on adaptive evolution of temperate and tropical lotus (Nelumbo nucifera), here we performed allele-specific expression (ASE) analyses on the tissues including flowers, leaves and rhizome from F1 hybrids of tropical and temperate lotus. For all investigated tissues in F1s, about 36% of genes showed ASE and about 3% of genes showed strong consistent ASE. Most of ASEs were biased towards the tropical parent in all surveyed samples, indicating that the tropical genome might be dominant over the temperate genome in gene expression of tissues from their F1 hybrids. We found that promoter sequences with similar allelic expression are more conserved than genes with significant or conditional ASE, suggesting the cis-regulatory sequence divergence underlie the allelic expression bias. We further uncovered biased genes being related to phenotypic differentiation between two lotus ecotypes, especially metabolic and phytohormone-related pathways in the rhizome. Overall, our study provides a global landscape of cis-regulatory variations between two lotus ecotypes and highlights their roles in rhizome growth variation for the climatic adaptation.

GRY79 encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice.

KEY MESSAGE: The green - revertible yellow79 mutant resulting from a single-base mutation suggested that the GRY79 gene encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. Functional studies of metallo-ß-lactamases and trihelix transcription factors in higher plants remain very sparse. In this study, we isolated the green-revertible yellow79 (gry79) mutant in rice. The mutant developed yellow-green leaves before the three-leaf stage but recovered to normal green at the sixth-leaf stage. Meanwhile, the mutant exhibited reduced level of chlorophylls and arrested development of chloroplasts in the yellow leaves. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene on rice chromosome 2. Map-based cloning revealed that the candidate gene was Os02g33610 encoding a putative metallo-ß-lactamase-trihelix chimera. In the gry79 mutant, a single-base mutation occurred in coding region of the gene, resulting in an amino acid change in the encoded protein. Furthermore, the mutant phenotype was rescued by transformation with the wild-type gene. Therefore, we have confirmed that the gry79 mutant phenotype resulted from a single-base mutation in GRY79 (Os02g33610) gene, suggesting that the gene encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. In addition, we considered that the gry79 mutant gene could be applicable as a leaf-color marker gene for efficient identification and elimination of false hybrids in commercial hybrid rice production.

Tissue Transglutaminase-Regulated Transformed Growth Factor-ß1 in the Parasite Links Schistosoma japonicum Infection with Liver Fibrosis.

Transforming growth factor (TGF-ß1) is among the strongest factors of liver fibrogenesis, but its association with Schistosoma-caused liver fibrosis is controversial. Tissue transglutaminase (tTG) is the principal enzyme controlling TGF-ß1 maturation and contributes to Sj-infected liver fibrosis. Here we aim to explore the consistency between tTG and TGF-ß1 and TGF-ß1 source and its correlation with liver fibrosis after Sj-infection. TGF-ß1 was upregulated at weeks 6 and 8 upon liver fibrosis induction. During tTG inhibition, TGF-ß1 level decreased in sera and liver of infected mice. TGF-ß1 showed positive staining in liver containing Sj adult worms and eggs. TGF-ß1 was also detected in Sj adult worm sections, soluble egg antigen and Sj adult worm antigen, and adult worms' culture medium. The TGF-ß1 mature peptide cDNA sequence and its extended sequence were amplified through RT-PCR and RACE-PCR using adult worms as template, and sequence is analyzed and loaded to NCBI GenBank (number GQ338152.1). TGF-ß1 transcript in Sj eggs was higher than in adult worms. In Sj-infected liver, transcriptional level of TGF-ß1 from Sj, but not mouse liver, correlated with liver fibrosis extent. This study provides evidence that tTG regulates TGF-ß1 and illustrates the importance of targeting tTG in treating Sj infection-induced fibrosis.

Characteristics of IL-17 induction by Schistosoma japonicum infection in C57BL/6 mouse liver.

Schistosomiasis japonica is a severe tropical disease caused by the parasitic worm Schistosoma japonicum. Among the most serious pathological effects of S. japonicum infection are hepatic lesions (cirrhosis and fibrosis) and portal hypertension. Interleukin-17 (IL-17) is a pro-inflammatory cytokine involved in the pathogenesis of many inflammatory and infectious conditions, including schistosomiasis. We infected C57BL/6 mice with S. japonicum and isolated lymphocytes from the liver to identify cell subsets with high IL-17 expression and release using flow cytometry and ELISA. Expression and release of IL-17 was significantly higher in hepatic lymphocytes from infected mice compared with control mice in response to both non-specific stimulation with anti-CD3 monoclonal antibody plus/anti-CD28 monoclonal antibody and PMA plus ionomycin. We then compared IL-17 expression in three hepatic T-cell subsets, T helper, natural killer T and γδT cells, to determine the major source of IL-17 during infection. Interleukin-17 was induced in all three subsets by PMA + ionomycin, but γδT lymphocytes exhibited the largest increase in expression. We then established a mouse model to further investigate the role of IL-17 in granulomatous and fibrosing inflammation against parasite eggs. Reducing IL-17 activity using anti-IL-17A antibodies decreased infiltration of inflammatory cells and collagen deposition in the livers of infected C57BL/6 mice. The serum levels of soluble egg antigen (IL)-specific IgGs were enhanced by anti-IL-17A monoclonal antibody blockade, suggesting that IL-17 normally serves to suppress this humoral response. These findings suggest that γδT cells are the most IL-17-producing cells and that IL-17 contributes to granulomatous inflammatory and fibrosing reactions in S. japonicum-infected C57BL/6 mouse liver.

Some characteristics of IL-5-producing T cells in mouse liver induced by Schistosoma japonicum infection.

Schistosome infection could cause significant liver damage in animal; Th2 cells play an important role in the progress of this disease. In our study, C57BL/6 mice were infected by Schistosoma japonicum and lymphocytes were isolated from the liver to detect some characteristics of interleukin-5 (IL-5)-producing T cells by different methods. The results revealed that S. japonicum infection could induce a large amount of IL-5 in mouse liver T cells by the means of fluorescent bead immunoassay and RT-PCR. Although, mouse liver contained many T cell subsets, such as Th cells, Tc cells, NKT cells, and γδ T cells. Fluorescence activated cell sorting results indicated that Th cells were the main source of IL-5 in the T cell population after phorbol 12-myristate 13-acetate and ionomycin stimulation. Moreover, the percentage of IL-5-producing Th cells continued to increase from 4 to 8 weeks after S. japonicum infection, which differed from the changes of IFN-γ(+) Th1 cells, IL-4(+) Th2 cells, and IL-17A(+) Th17 cells during S. japonicum infection. Additionally, cytokines co-expression results demonstrated that 36.2 % of IL-5(+) Th cells could express IL-4, and 10 % of it could produce IFN-γ or IL-17A. Collectively, these findings implied that IL-5-producing Th cells posses some properties which differ from other cytokines secreting Th cells.

Pausing behavior of late bilingual and monolingual English speakers.

This study examined the differences in the pausing behavior between native and non-native English speakers. Specifically, it examined the location and duration of pauses in relation to the syntactic and lexical complexity of the clauses in which these pauses occur and the nature of the prosodic phrasing of the utterances containing pauses. Speech samples from 10 native (L1) English and 10 Mandarin non-native English speakers from the Archive of L1 and L2 Scripted and Spontaneous Transcripts and Recordings (ALLSSTAR) were included in the analysis. The results showed that lower-level prosodic boundaries and syntactically complex phrases were associated with significantly longer pause duration in the L2 speech. Additionally, phrases with less frequent words tended to induce longer pauses. These findings suggest that insufficient knowledge of the L2 syntax, lexicon, and prosody might determine the location and duration of pauses and ultimately affect the speech fluency of L2 speakers.

Characteristics of Th9 cells in Schistosoma japonicum-infected C57BL/6 mouse mesenteric lymph node.

Interleukin 9 (IL-9) is an effective cytokine secreted by newly defined Th9 cells, which is involved in allergic and infectious diseases. In this study, lymphocytes were isolated from mesenteric lymph node (MLN), spleen, liver, lung, and Peyer's patches (PP) of C57BL/6 mice 5-6 weeks after S. japonicum infection, intracellular cytokine staining was done to detect the percentage of IL-9-producing CD4+ T cells. The qPCR and ELISA were used to verify the content of IL-9 in MLN. The population of IL-9-producing lymphocyte subset was identified by FACS. In addition, the dynamic changes and cytokine profiles of Th9 cells in the MLN of infected mice were detected by FACS. ELISA was used to detect IL-9 induced by soluble egg antigen (SEA) from isolated lymphocytes in mouse MLN. The results showed that the percentage of IL-9-secreting Th9 cells in the MLN of the infected mouse was higher than that in the spleen, liver, lung, or PP. Though CD8+ Tc cells, NKT cells, and γδT cells could secrete IL-9, CD4+ Th cells were the main source of IL-9 in S. japonicum-infected C57BL/6 mice (P < 0.05). The percentage of Th9 cells in MLN of infected mouse increased from week 3-4, and reached a peak at week 5-6, then began to decrease from week 7-8 (P < 0.05). Moreover, Th9 cells could also secrete a small amount of IL-4, IFN-γ, IL-5, and IL-10. Our results suggested a higher percentage of Th9 cells was induced in the MLN of S. japonicum-infected mice, which might play an important role in the early stage of S. japonicum-induced disease.

[Immune response of Th17 cells in mesenteric lymph node of mice infected by Schistosoma japonicum].

OBJECTIVE: To observe the immune response of Th17 cells in mesenteric lymph node (MLN) of C57BL/6 mice infected by Schistosoma japonicum. METHODS: Twenty C57BL/6 mice were randomly divided into infected group and control group each with ten mice. The mice in infected group were infected each with 40 +/- 5 S. japonicum cercariae. Five to six weeks later, MLN lymphocytes were separated and stimulated for 4 h by anti-CD3 (1 microg/ml) and anti-CD28 (1 microg/ml) before examination of IL-17 and retinoic acid receptor-related orphan receptor gammat (ROR-gammat) mRNA by reverse transcription PCR. The level of IL-17 and IFN-gamma was detected by ELISA after culturing with supernatant for 72h. MLN lymphocytes were stimulated for 5h by 10 ng/ml phorbol myristoyl acetate (PMA) and 1 microg/ml ionomycin. The intracellular cytokines were stained and the content of Th17 and other cytokines was examined by flow cytometry. RESULTS: The level of IFN-gamma [(214.3 +/- 62.6) pg/ml] and IL-17 [(176.8 +/- 62.1) pg/ml] in the supernatant of cultured MLN cells from the infected mice was significantly higher than that of normal mice [(467 +/- 13.9) and 0 pg/ml) (P < 0.05). The expression level of IL-17 and ROR-gammat mRNA was also considerably higher than that of normal mice. IL-17+ IL-4+, IL-17+ IFN-gamma+, IL-17+ IL-5+ and IL-17+ IL-9 cells accounted for 0.06%, 0.02%, 0.02%, and 0.01% of the mesenteric lymph node CD4+ T cells of the infected mice, respectively. However, IL-17+ IL-10+ and IL-17+ Foxp3+ cells were undetected. CONCLUSION: The MLN of S. japonicum-infected C57BL/6 mice can induce the production of Th17 cells, and these cells can secrete IL-4, less IFN-gamma, IL-5 and IL-9, but not IL-10, and can not express Foxp3 in the infected mice.

Genome-wide association study of traits in sacred lotus uncovers MITE-associated variants underlying stamen petaloid and petal number variations.

Understanding the genetic variants responsible for floral trait diversity is important for the molecular breeding of ornamental flowers. Widely used in water gardening for thousands of years, the sacred lotus exhibits a wide range of diversity in floral organs. Nevertheless, the genetic variations underlying various morphological characteristics in lotus remain largely unclear. Here, we performed a genome-wide association study of sacred lotus for 12 well-recorded ornamental traits. Given a moderate linkage disequilibrium level of 32.9 kb, we successfully identified 149 candidate genes responsible for seven flower traits and plant size variations, including many pleiotropic genes affecting multiple floral-organ-related traits, such as NnKUP2. Notably, we found a 2.75-kb presence-and-absence genomic fragment significantly associated with stamen petaloid and petal number variations, which was further confirmed by re-examining another independent population dataset with petal number records. Intriguingly, this fragment carries MITE transposons bound by siRNAs and is related to the expression differentiation of a nearby candidate gene between few-petalled and double-petalled lotuses. Overall, these genetic variations and candidate genes responsible for diverse lotus traits revealed by our GWAS highlight the role of transposon variations, particularly MITEs, in shaping floral trait diversity.

3D hollow Bi2O3@CoAl-LDHs direct Z-scheme heterostructure for visible-light-driven photocatalytic ammonia synthesis.

In this paper, the novel 3D hollow Z-scheme heterojunction photocatalysts based on Bi2O3 and CoAl layered double hydroxides (Bi2O3@CoAl-LDHs) were prepared for efficient visible-light-driven photocatalytic ammonia synthesis. The synthesized nanohybrid exhibits excellent photocatalytic ammonia synthesis performance (48.7 µmol·L-1·h-1) and structural stability, which is primarily attributed to the fact that Z-scheme heterojunction significantly enhanced lifetime of photogenerated carriers (6.22 ns) and transfer efficiency of surface photogenerated electrons (72.5%). Strict control experiments and nitrogen isotope labeling results show that nitrogen and hydrogen in the produced ammonia come from nitrogen and water in the reactant respectively. Electron paramagnetic resonance (EPR) experiments and density functional theory (DFT) calculations further reveal that the built-in electric field due to the difference between Bi2O3 and CoAl-LDHs is the key to constructing the Z-scheme heterojunction. In addition, results of partial density of states (PDOS) show that Co in Bi2O3@CoAl-LDHs composite is the active site for photocatalytic N2 fixation.

Nelumbo genome database, an integrative resource for gene expression and variants of Nelumbo nucifera.

Sacred lotus (Nelumbo nucifera, or lotus) is one of the most widely grown aquatic plant species with important uses, such as in water gardening and in vegetable and herbal medicine. A public genomic database of lotus would facilitate studies of lotus and other aquatic plant species. Here, we constructed an integrative database: the Nelumbo Genome Database (NGD, http://nelumbo.biocloud.net ). This database is a collection of the most updated lotus genome assembly and contains information on both gene expression in different tissues and coexpression networks. In the NGD, we also integrated genetic variants and key traits from our 62 newly sequenced lotus cultivars and 26 previously reported cultivars, which are valuable for lotus germplasm studies. As applications including BLAST, BLAT, Primer, Annotation Search, Variant and Trait Search are deployed, users can perform sequence analyses and gene searches via the NGD. Overall, the valuable genomic resources provided in the NGD will facilitate future studies on population genetics and molecular breeding of lotus.

A cross-sectional study: Comparing the attitude and knowledge of medical and non-medical students toward 2019 novel coronavirus.

BACKGROUND AND OBJECTIVES: Since December 2019, the rapid epidemic spread of COVID-19 in China has aroused the attention of the government and the public. The purpose of this study is to investigate the attitude and knowledge among medical students and non-medical students toward SARS-CoV-2 infection. METHODS: A web-based survey was disseminated to the students from medical colleges and comprehensive universities via the survey website (www.wjx.cn) and via WeChat. Participation in the study was voluntary with the instruction to click on the website or scan the QR code to complete the anonymous electronic questionnaire from February 5 to 7, 2020. RESULTS: The questionnaire was completed by 588 students from 20 colleges and universities in China. Of the respondents, 66.0% were medical students and 34.0% were non-medical students. 99.6 % of the students held an optimistic attitude toward the COVID-19 epidemic situation. The majority of participants had a good level of knowledge of common symptoms, transmission, and prevention of the disease. In a comparison between non-medical students with medical students, the medical students had a deeper understanding of COVID-19. In this study, we also found that female students had a better understanding of transmission and prevention than male students did. CONCLUSIONS: The majority of students who participated in the questionnaire had a positive attitude and a good perception about COVID-19.

Highly Sensitive Detection of Melamine Based on the Fluorescence Resonance Energy Transfer between Conjugated Polymer Nanoparticles and Gold Nanoparticles.

Adding melamine as additives in food products will lead to many diseases and even death. However, the present techniques of melamine detection require time-consuming steps, complicated procedures and expensive analytical apparatus. The fluorescent assay method was facile and highly sensitive. In this work, a fluorescence resonance energy transfer (FRET) system for melamine detection was constructed based on conjugated polymer nanoparticles (CPNs) and gold nanoparticles (AuNPs). The energy transfer efficiency is up to 82.1%, and the system is highly selective and sensitive to melamine detection with a lower detection limit of 1.7 nmol/L. Moreover, the interaction mechanism was explored. The results showed that the fluorescence of CPNs were firstly quenched by AuNPs, and then restored after adding melamine because of reducing FRET between CPNs and AuNPs. Lastly, the proposed method was carried out for melamine detection in powdered infant formula with satisfactory results.