Identification of genetic causes in children with unexplained epilepsy based on trio-whole exome sequencing.

Genotype and clinical phenotype analyses of 128 children were performed based on whole exome sequencing (WES), providing a reference for the provision of genetic counseling and the precise diagnosis and treatment of epilepsy. A total of 128 children with unexplained epilepsy were included in this study, and all their clinical data were analyzed. The children's treatments, epilepsy control, and neurodevelopmental levels were regularly followed up every 3 months. The genetic diagnostic yield of the 128 children with epilepsy is 50.8%, with an SNV diagnostic yield of 39.8% and a CNV diagnostic yield of 12.5%. Among the 128 children with epilepsy, 57.0% had onset of epilepsy in infancy, 25.8% have more than two clinical seizure forms, 62.5% require two or more anti-epileptic drug treatments, and 72.7% of the children have varying degrees of psychomotor development retardation. There are significant differences between ages of onset, neurodevelopmental levels and the presence of drug resistance in the genetic diagnostic yield (all p < 0.05). The 52 pathogenic/likely pathogenic SNVs involve 31 genes, with genes encoding ion channels having the largest number of mutations (30.8%). There were 16 cases of pathogenic/possibly pathogenic CNVs, among which the main proportions of CNVs were located in chromosome 15 and chromosome 16. Trio-WES is an essential tool for the genetic diagnosis of unexplained epilepsy, with a genetic diagnostic yield of up to 50.8%. Early genetic testing can provide an initiate appropriate therapies and accurate molecular diagnosis.

A de novo gene originating from the mitochondria controls floral transition in Arabidopsis thaliana.

De novo genes created in the plant mitochondrial genome have frequently been transferred into the nuclear genome via intergenomic gene transfer events. Therefore, plant mitochondria might be a source of de novo genes in the nuclear genome. However, the functions of de novo genes originating from mitochondria and the evolutionary fate remain unclear. Here, we revealed that an Arabidopsis thaliana specific small coding gene derived from the mitochondrial genome regulates floral transition. We previously identified 49 candidate de novo genes that induce abnormal morphological changes on overexpression. We focused on a candidate gene derived from the mitochondrial genome (sORF2146) that encodes 66 amino acids. Comparative genomic analyses indicated that the mitochondrial sORF2146 emerged in the Brassica lineage as a de novo gene. The nuclear sORF2146 emerged following an intergenomic gene transfer event in the A. thaliana after the divergence between Arabidopsis and Capsella. Although the nuclear and mitochondrial sORF2146 sequences are the same in A. thaliana, only the nuclear sORF2146 is transcribed. The nuclear sORF2146 product is localized in mitochondria, which may be associated with the pseudogenization of the mitochondrial sORF2146. To functionally characterize the nuclear sORF2146, we performed a transcriptomic analysis of transgenic plants overexpressing the nuclear sORF2146. Flowering transition-related genes were highly regulated in the transgenic plants. Subsequent phenotypic analyses demonstrated that the overexpression and knockdown of sORF2146 in transgenic plants resulted in delayed and early flowering, respectively. These findings suggest that a lineage-specific de novo gene derived from mitochondria has an important regulatory effect on floral transition.

Integration of metabolomics and transcriptomics analyses reveals sphingosine-1-phosphate-mediated S1PR2/PI3K/Akt pathway involved in Talaromyces marneffei infection of macrophages.

Talaromycosis is a fatal mycosis caused by the thermally dimorphic fungus Talaromyces marneffei (T. marneffei). The pathogenic mechanisms of talaromycosis are still poorly understood. This work combined metabolomics, transcriptomics, and verification experiments in vivo and in vitro to detect metabolic profiles and differentially expressed genes (DEGs) in T. marneffei infected and uninfected macrophages to explore possible pathogenesis and underlying mechanisms. A total of 256 differential metabolites (117 up-regulated and 148 down-regulated) and 1320 DEGs (1286 up-regulated and 34 down-regulated) were identified between the two groups. Integrative metabolomics and transcriptomics analysis showed sphingolipid signaling pathway is the most influential. Verification experiments showed that compared with the control group, the production of sphingosine-1-phosphate (S1P) and the expression of the S1PR1, S1PR2, phosphor-PI3K, and phosphor-Akt genes involved in the sphingolipid signaling pathway have significantly increased in the T. marneffei infection group (p < 0.05). T. marneffei activates the S1PR2/PI3K/Akt pathways in J774A.1 macrophage, regulation of the S1P singling might serve as a promising therapeutic strategy for talaromycosis.

Effect of small coding genes on the circadian rhythms under elevated CO2 conditions in plants.

Increase in atmospheric carbon dioxide (CO2) has a significant effect on plant growth and development. To explore the elevated-CO2 response, we generated transcriptional profiles over a time course (2 h-14 days) of exposure to elevated CO2 in Arabidopsis thaliana. Genes related to photosynthesis were down-regulated and circadian rhythm-related genes were abnormally regulated in the early to middle phase of elevated CO2 exposure. To understand the novel mechanism of elevated CO2 signaling, we focused on 42 unknown small coding genes that showed differential expression patterns under elevated CO2 conditions. Four transgenic plants overexpressing the small coding gene exhibited a growth-defective phenotype under elevated CO2 but not under current CO2. Transcriptome analysis showed that circadian rhythm-related genes were commonly regulated in four transgenic plants. These circadian rhythm-related genes were transcribed in the dark when CO2 concentrations in the leaf was high. Taken together, our identified four small coding genes are likely to participate in elevated CO2 signaling to the circadian rhythm.

[Effects of rapamycin on glucose-induced autophagy and apoptosis of corpus cavernosum smooth muscle cells in SD rats].

OBJECTIVE: To observe the effects of rapamycin on glucose-induced autophagy and apoptosis of corpus cavernosum smooth muscle cells (CCSMC) in SD rats. METHODS: After primary isolation, purification and identification, CCSMCs were treated with glucose at 5.6, 10, 20, 30 and 40 mmol/L for 48 hours. The autophagy flow was detected in the cells transfected with mCherry-GFP-LC3B double fluorescent adenovirus, the protein and mRNA expressions of autophagy-related gene 5 (ATG5), microtubule-associated protein 1 light chain 3 (LC3), P62 and Beclin-1 determined by Western blot and PCR and the apoptosis of the CCSMC measured by flow cytometry, followed by detection of the changes in the protein and mRNA levels of LC3, Beclin-1 and P62 and the apoptosis of the cells after 2-hour pretreatment with 600 nmol/L rapamycin and then 48-hour treatment with 40 mmol/L glucose. RESULTS: Autophagy was observed in the CCSMCs treated with different concentrations of glucose, the strongest in the 10 mmol/L group (P < 0.05) and then gradually decreasing with the increase of glucose concentration (P < 0.05). The apoptosis rate of the CCSMCs was remarkably increased in a concentration-dependent manner after treated with glucose (P < 0.01) though with no statistically significant difference between the 5.6 and 10 mmol/L groups (P = 0.302). After pretreatment with rapamycin, the expression of the LC3 protein and those of the Beclin-1 protein and mRNA were markedly up-regulated (P < 0.05), while those of the P62 protein and mRNA remarkably down-regulated (P < 0.05), indicating the enhancement of autophagy, and the apoptosis rate of the CCSMCs dramatically decreased (P < 0.01). CONCLUSIONS: In vitro culture of CCSMCs with glucose within a certain range of concentration can promote autophagy, inhibit the apoptosis and thus have a protective effect on the cells, but when the proper concentration exceeded, it may also reduce autophagy, promote the apoptosis and consequently affect the vitality and function of CCSMCs.

[Quantitative analysis of fermented aerial part of Bupleurum chinense and prediction of their antimicrobial activity].

The aim of this study was to predict the anti-microbial components in the aerial part of Bupleurum chinense fermented by Lactobacillus plantarum through analyzing the correlation between contents of bioactive components and their inhibitory action for pathogenic bacteria. In this study, the UPLC-MS-MS detection method was established for eight flavonoids(kaempferol-3-O-ß-D-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, iridin, quercetin-3-O-ß-L-arabinoside, kaempferol) and DL-3-phenyllactic acid, and the dynamic change of their contents at fermentation course were monitored. Meanwhile, the experiment employed five common no-naquatic pathogenic bacteria(Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus subtilis), and four common aquatic pathogenic bacteria(Aeruginosa hydrophila, Delayed Edwards, Vibrio alginolyticus, and Vibrio harveyi) to validate in vitro anti-microbial activity of the fermented aerial part of B. chinense at different fermentation time points. Finally, the Pearson correlation analysis was applied to predict the anti-microbial components of the fermented aerial part of B. chinense.The established UPLC-MS-MS method showed a good linearity and the widest linear range was from 0.19 µg·mL~(-1) to 50 µg·mL~(-1). The limit of quantitation and the limit of detection were 0.19-1.56 µg·mL~(-1) and 0.1-0.78 µg·mL~(-1) respectively. During the fermentation within 48 h, the contents of three flavonoids(rutin, quercetin-3-O-ß-L-arabinoside, isoquercitrin) and DL-3-phenyllactic acid from the fermented aerial part of B. chinense increased sharply. In the process of fermentation, the anti-microbial effect of the fermented aerial part of B. chinense on aquatic pathogens was significantly stronger than that on non-aquatic pathogens. Furthermore, Pearson correlation analysis predicted that isoquercitrin, rutin, quercetin-3-O-ß-L-arabinoside and DL-3-phenyllactic acid showed significant correlation with the four aquatic pathogens. This study revealed that the fermented aerial part of B. chinense had a high sensitivity to aquatic pathogens, which may be caused by the increased contents of isoquercitrin, rutin, quercetin-3-O-ß-L-arabinoside and DL-3-phenyllactic acid. In conclusion, this study provides a theoretical basis and new idea for the further development of the large amount of wasteful aerial part of Bupleurum chinense.

[Effects of autophagy on spermatogenesis and sperm motility].

Decreased sperm count, low sperm motility and sperm malformation are important factors of male infertility, but their pathogenic mechanisms have not yet been elucidated. Autophagy is an evolutionarily highly conservative cellular process and plays an important role in spermatogenic cells in both normal physiological and adverse conditions. On the one hand, autophagy can degrade the majority of long-lived proteins and organelles to maintain the intracellular homeostasis of spermatogenic cells, the meiosis of spermatocytes and spermiogenesis, and improve sperm motility. On the other hand, excessive autophagy can lead to excessive consumption of proteins and damage to organelles, induce cellular dysfunction, and result in sperm count reduction, spermatogenic defects, and low sperm motility. This review presents an overview of the recent studies on the influences of autophagy on spermatogenesis and sperm motility, aiming for some new therapeutic targets for male infertility.

Programming of Plant Leaf Senescence with Temporal and Inter-Organellar Coordination of Transcriptome in Arabidopsis.

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

Relationship Between Human mutL Homolog 1 (hMLH1) Hypermethylation and Colorectal Cancer: A Meta-Analysis.

BACKGROUND Hypermethylation of CpG islands in gene promoter regions is an important mechanism of gene inactivation in cancers. Promoter hypermethylation of human mutL homolog 1 (hMLH1) has been implicated in a subset of colorectal cancers that show microsatellite instability (MSI), while the connection of the epigenetic inactivation of hMLH1 in colorectal cancers remains unknown. The aim of this study was to evaluate the relationship between the promoter hypermethylation of hMLH1 and colorectal cancers by performing a meta-analysis. MATERIAL AND METHODS Eligible studies were identified through searching PubMed, Cochrane Library, Web of Science, and Google Scholar databases. R Software including meta packages was used to calculate the pooled and odds ratios (ORs) with corresponding confidence intervals (CIs). Funnel plots were also performed to evaluate publication bias. RESULTS This meta-analysis obtained 45 articles, including 4096 colorectal cancer patients, and identified a significant association between hMLH1 hypermethylation and colorectal cancer risk using the fixed-effects model (OR=8.3820; 95% CI, 6.9202~10.1527; z=21.7431; P<0.0001) and random effects model pooled (OR=10.0963; 95% CI, 6.1919~16.4626; z=9.2688; P<0.0001). The significant relationship was found in subgroup analyses. CONCLUSIONS The results of this meta-analysis show a significant association between hMLH1 hypermethylation and colorectal cancer risk.

Research progress on genetic heterogeneity between primary and paired metastatic colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in China. A standard practice in treating metastatic CRC (mCRC) is to predict benefits of the anti-EGFR monoclonal antibody treatment based on the somatic mutation spectrum. Because metastatic samples are difficult to obtain clinically, primary tumors are normally used instead. However, due to the genetic heterogeneity between primary and paired metastatic tumors, primary site biopsy always underrepresents the mutational landscape of metastatic sites. Currently, the extent of genetic heterogeneity between primary and paired mCRC is still under debate. Here, we review comparative studies of primary and matched mCRC and discuss the underlying causes and potential strategies.

Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis.

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.

The role of lipid peroxidation in epithelial-mesenchymal transition of retinal pigment epithelial cells.

Epithelial-Mesenchymal Transition (EMT) of retinal pigment epithelial (RPE) cells is recognized as pivotal in various retinal diseases. Previous studies have suggested a reciprocal regulation between reactive oxygen species (ROS) and EMT, though the involvement of peroxidized lipids or the effects of reducing them has remained unclear. The present study disclosed that EMT of ARPE-19 cells induced by TGF-ß2 and TNF-α involves increased lipid peroxidation, and Ferrostatin-1 (Fer-1), a lipophilic antioxidative agent, successfully inhibited the increase in lipid peroxidation. Fer-1 suppressed the formation of EMT-associated fibrotic deposits, while EMT induction or Fer-1 treatment did not influence the cell viability or proliferation. Functionally, Fer-1 impeded EMT-driven cell migration and reduction in transepithelial electrical resistance. It demonstrated regulatory prowess by downregulating the mesenchymal marker fibronectin, upregulating the epithelial marker ZO-1, and inhibiting the EMT-associated transcriptional factor ZEB1. Additionally, VEGF, a major pathogenic cytokine in various retinal diseases, is also upregulated during EMT, and Fer-1 significantly mitigated the effect. The present study disclosed the involvement of lipid peroxidation in EMT of RPE cells, and suggests the suppression of lipid peroxidation may be a potential therapeutic target in retinal diseases in which EMT is implicated.

Identification of gene signatures and molecular mechanisms underlying the mutual exclusion between psoriasis and leprosy.

Leprosy and psoriasis rarely coexist, the specific molecular mechanisms underlying their mutual exclusion have not been extensively investigated. This study aimed to reveal the underlying mechanism responsible for the mutual exclusion between psoriasis and leprosy. We obtained leprosy and psoriasis data from ArrayExpress and GEO database. Differential expression analysis was conducted separately on the leprosy and psoriasis using DEseq2. Differentially expressed genes (DEGs) with opposite expression patterns in psoriasis and leprosy were identified, which could potentially involve in their mutual exclusion. Enrichment analysis was performed on these candidate mutually exclusive genes, and a protein-protein interaction (PPI) network was constructed to identify hub genes. The expression of these hub genes was further validated in an external dataset to obtain the critical mutually exclusive genes. Additionally, immune cell infiltration in psoriasis and leprosy was analyzed using single-sample gene set enrichment analysis (ssGSEA), and the correlation between critical mutually exclusive genes and immune cells was also examined. Finally, the expression pattern of critical mutually exclusive genes was evaluated in a single-cell transcriptome dataset. We identified 1098 DEGs in the leprosy dataset and 3839 DEGs in the psoriasis dataset. 48 candidate mutually exclusive genes were identified by taking the intersection. Enrichment analysis revealed that these genes were involved in cholesterol metabolism pathways. Through PPI network analysis, we identified APOE, CYP27A1, FADS1, and SOAT1 as hub genes. APOE, CYP27A1, and SOAT1 were subsequently validated as critical mutually exclusive genes on both internal and external datasets. Analysis of immune cell infiltration indicated higher abundance of 16 immune cell types in psoriasis and leprosy compared to normal controls. The abundance of 6 immune cell types in psoriasis and leprosy positively correlated with the expression levels of APOE and CYP27A1. Single-cell data analysis demonstrated that critical mutually exclusive genes were predominantly expressed in Schwann cells and fibroblasts. This study identified APOE, CYP27A1, and SOAT1 as critical mutually exclusive genes. Cholesterol metabolism pathway illustrated the possible mechanism of the inverse association of psoriasis and leprosy. The findings of this study provide a basis for identifying mechanisms and therapeutic targets for psoriasis.

Early results of the integrative epigenomic-transcriptomic landscape of colorectal adenoma and cancer.

BACKGROUND: Aberrant methylation is common during the initiation and progression of colorectal cancer (CRC), and detecting these changes that occur during early adenoma (ADE) formation and CRC progression has clinical value. AIM: To identify potential DNA methylation markers specific to ADE and CRC. METHODS: Here, we performed SeqCap targeted bisulfite sequencing and RNA-seq analysis of colorectal ADE and CRC samples to profile the epigenomic-transcriptomic landscape. RESULTS: Comparing 22 CRC and 25 ADE samples, global methylation was higher in the former, but both showed similar methylation patterns regarding differentially methylated gene positions, chromatin signatures, and repeated elements. High-grade CRC tended to exhibit elevated methylation levels in gene promoter regions compared to those in low-grade CRC. Combined with RNA-seq gene expression data, we identified 14 methylation-regulated differentially expressed genes, of which only AGTR1 and NECAB1 methylation had prognostic significance. CONCLUSION: Our results suggest that genome-wide alterations in DNA methylation occur during the early stages of CRC and demonstrate the methylation signatures associated with colorectal ADEs and CRC, suggesting prognostic biomarkers for CRC.

Network Pharmacology Revealed the Mechanisms of Action of Lithospermum erythrorhizon Sieb on Atopic Dermatitis.

Aim: The application of network analysis algorithms promoted the development of network pharmacology. This study aimed to combine network pharmacology and signed random walk with restart (SRWR) to reveal the mechanism by which Lithospermum erythrorhizon Sieb (LES) exerts effects on atopic dermatitis (AD). Methods: The compounds and targets of LES were retrieved from Traditional Chinese Medicine Integrated Database (TCMID) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and important compounds and targets were identified by intersection analysis and protein-protein interaction (PPI) network. Results: We found that active LES-derived compounds such as caffeic acid, Isovaleric acid, Arnebinol, and Alannan may inhibit PTGS2, HSP90AA1 and MAPK14, which are key mediators involved in PI3K-Akt pathway, vascular endothelial growth factor signaling pathway, Fc epsilon RI signaling pathway, and calcium signaling pathway. Conclusion: The application of SRWR could identify potential targets of LES with a low false-positive rate and help elucidate the mechanism of action of traditional Chinese medicine.

Corrigendum: IL36G is associated with cutaneous antiviral competence in psoriasis.

[This corrects the article DOI: 10.3389/fimmu.2022.971071.].

L36G is associated with cutaneous antiviral competence in psoriasis.

Background: Psoriasis is a common inflammatory skin disease that has a great impact on patients' physical and mental health. However, the causes and underlying molecular mechanisms of psoriasis are still largely unknown. Methods: The expression profiles of genes from psoriatic lesion samples and skin samples from healthy controls were integrated via the sva software package, and differentially expressed genes (DEGs) between psoriasis and healthy skin were screened by the limma package. Furthermore, GO and KEGG pathway enrichments for the DEGs were performed using the Clusterprofiler package. Protein-protein interaction (PPI) networks for the DEGs were then constructed to identify hub genes. scGESA analysis was performed on a single-cell RNA sequencing dataset via irGSEA. In order to find the cytokines correlated with the hub genes expression, single cell weighted gene co-expression network analyses (scWGCNA) were utilized to build a gene co-expression network. Furthermore, the featured genes of psoriasis found in suprabasal keratinocytes were intersected with hub genes. We then analyzed the expression of the intersection genes and cytokines in the integrated dataset. After that, we used other datasets to reveal the changes in the intersection genes' expression levels during biological therapy. The relationship between intersection genes and PASI scores was also explored. Results: We identified 148 DEGs between psoriatic and healthy samples. GO and KEGG pathway enrichment analysis suggested that DEGs are mainly involved in the defense response to other organisms. The PPI network showed that 11 antiviral proteins (AVPs) were hub genes. scGSEA analysis in the single-cell transcriptome dataset showed that those hub genes are highly expressed in keratinocytes, especially in suprabasal keratinocytes. ISG15, MX1, IFI44L, and IFI27 were the characteristic genes of psoriasis in suprabasal keratinocytes. scWGCNA showed that three cytokines-IL36G, MIF, and IL17RA-were co-expressed in the turquoise module. Only interleukin-36 gamma (IL36G) was positively correlated with AVPs in the integrated dataset. IL36G and AVPs were found co-expressed in a substantial number of suprabasal keratinocytes. Furthermore, we found that the expression levels of IL36G and the 4 AVPs showed positive correlation with PASI score in patients with psoriasis, and that these levels decreased significantly during treatment with biological therapies, but not with methotrexate. Conclusion: IL36G and antiviral proteins may be closely related with the pathogenesis of psoriasis, and they may represent new candidate molecular markers for the occurrence and severity of psoriasis.

Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter.

The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.

Ubiquitin promoter-terminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce.

Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.

Evaluation of influence of acupuncture and electro-acupuncture for blood perfusion of stomach by laser Doppler blood perfusion imaging.

The objective of this study is to observe effects of acupuncture and electro-acupuncture (EA) on blood perfusion in the stomach, and probe into the application of laser Doppler blood perfusion imaging technique in the study of the effect of acupuncture and moxibustion on the entrails. In the acupuncture group of 20 rats, acupuncture was given at "Zusanli" (ST 36) and in EA group of 18 rats, EA was applied at "Zusanli" (ST 36), with 18 rats without acupuncture used as control group. Changes of blood perfusion and microcirculation distribution in the stomach were investigated with laser Doppler blood perfusion imager (LDPI). The laser Doppler blood perfusion image could clearly display changes of blood flow distribution in the stomach before and after acupuncture. After acupuncture or EA was given at "Zusanli" (ST 36), the blood perfusion in the stomach increased significantly, the blood perfusion in the blood vessels and microcirculation of other parts significantly increased, and the maximum increase of the blood perfusion was found at 10 min after acupuncture or EA, with increases of 0.50 _ 0.11 (PU) and 0.66 _ 0.16 (PU), respectively, and the blood perfusion still kept at a higher degree within 10 min after ceasing of the acupuncture or EA. While the blood perfusion in the stomach in the rat of the control group tended to gradual decrease. It has been concluded that both acupuncture and EA can increase blood perfusion in the stomach, the EA having stronger action, and LDPI can display the regulative action of acupuncture on the blood vessel of the stomach by using an image.