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Introduction Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Artemisia annuais an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression ofProduction of Anthocyanin Pigment 1(PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences ofA. annuaand obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-Box cis-element. Eight promoters were cloned for activity analysis. Duel luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 μM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per petri dish. HPLC-qTOF-MS/MS based profiling showed that these concentrations did not alter anthocyanin profiles. Real time RT-PCR was completed to characterize the expressionPAP1and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutivePAP1transgene and three pathway genes significantly and eliminated chalcone synthase gene in expression either significantly or slightly. These data indicate that the constitutivePAP1expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells ofA. annua.ConclusionPromoters of eight anthocyanin pathway genes were cloned. Four types of AC cis-elements were identified from six promoters and G-Box elements were also determined from six promoters. PAP1 enhanced the activity of eight promoters. 2,4-D downregulated the expression of the constitutive PAP1 transgene leading to the decrease the biosynthesis of anthocyanins.

Artemisia annuais the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs),; however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated fromA. annua. In this study, our goals were to engineer anthocyanins inA. annuacells and to understand the artemisinin biosynthesis in anthocyanin-producing cells. ArabidopsisPAP1(AtPAP1) was used to engineer four types of transgenic anthocyanin-producingA. annua(TAPA1 to 4) cells. Three wild type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from DHAA in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells ofA. annua.

The computer-generated hologram (CGH) enables the ultra-high accuracy of surface measurement but causes the wavefront degeneration in the optical system. In this article, we give a high-accuracy analytical simulation of the wavefront degeneration in null test by the elliptical Gaussian model. We propose an analytical expression of instrumental transfer function (ITF) for the CGH null test without knowing the phase distribution of CGH, which gives an efficient instruction to suppress the wavefront degeneration. The ITF of the interferometric null test for a ∅3m aspheric mirror can be optimized from 0 to 0.65 at 0.4 Nyquist frequency.

To examine the effects of i.v. administration of MK-571, a MRP4/5 pump inhibitor, on urethral function in the urethane-anesthetized rat, and the changes of urethral multidrug resistance protein 5 (MRP5) pump in streptozotocin (STZ)-induced diabetes mellitus (DM) rats.Isovolumetric cystometry and urethral perfusion pressure (UPP) measurements were carried out in normal control (NC) group and 8week DM groups under urethane anesthesia. When stable rhythmic bladder contractions were showed, UPP parameters were recorded after successive administration of various dose of MK-571. Additionally, urethral cyclic guanosine monophosphate (cGMP) protein level was evaluated by ELISA, and changes of MRP5 pump and neurogenic nitric oxide synthase (nNOs) in the urethra were examined with immunohistochemical staining and Western blot analysis.In NC group, UPPMK-571 could not restore NO-mediated urethral relaxation dysfunction in DM rats, which may be attributed to the up-regulation of urethral MRP5 pump, and thus decrease of intracellular cGMP concentration in the urethra. These novel results would be useful for a better understanding of DM-related lower urinary tract dysfunction LUT (LUTD). Also, they could be helpful to study the importance of MRP pumps in the control of urethral relaxation mechanisms under physiological and pathological states.

Although much is known about how adipose tissue affects the development of clear cell renal carcinoma (ccRCC), little information is available for the utility of sex-specific abdominal visceral fat composition as a predictor of clear cell renal carcinoma (ccRCC) T stage. We conducted CT-based sex-specific abdominal fat measurements in ccRCC patients to assess whether VFA distribution could predict the ccRCC T stage. In total, 253 patients (182 males and 71 females) from our hospital with pathologically confirmed ccRCC (178 low T-stage and 75 high T-stage) were retrospectively reviewed for the present study. Computed tomography (CT) scans were assessed using ImageJ to differentiate between the visceral and subcutaneous fat areas (VFA and SFA), after which the relative VFA (rVFA) and total fat area (TFA) were computed. The relationships between these fat area-related variables, patient age, sex, and BMI, and ccRCC T stage were then evaluated through univariate and multivariate logistic regression analysis to clarify the association between general or sex-specific abdominal visceral fat and T stage. Following adjustment for age, males with high T stage ccRCC exhibited an increased rVFA as compared to males with low T stage ccRCC, with the same relationship being observed among females. This association between rVFA and high T stage was confirmed through both univariate and multivariate models. As thus, sex-specific visceral fat composition is a reliable independent predictor that can identify both male and female patients with high T stage ccRCC.

Purpose: To identify the value of prostate-specific antigen density (PSAD) and prostate-specific antigen density of the transition zone (PSADTZ) in improving the sensitivity and specificity of the prostate multiparameter magnetic resonance imaging (mp-MRI), for the purpose of predicting prostate cancer (PCa) and grade reclassification in men with prostate-specific antigen (PSA) between 4 and 20 ng/mL to reduce unnecessary prostate biopsies. Patients and Methods: Between 2018 and 2020, we retrospectively identified 283 consecutive men in Shanghai Jiao Tong University Affiliated Sixth People’s Hospital who had mp-MRI and PSA test within 3 months before prostate biopsies. Total prostate volume (TPV) and transition zone volume (TZV) were measured on mp-MRI. PSA, PSAD, and PSADTZ were compared to improve the sensitivity and specificity of positive biopsy cores and pathological stage by univariate analyses and through the receiver operating curve (ROC). We were focused primarily on the MRI-positive patients with PSA levels of 4-20ng/ml who were most likely subjected to unnecessary repeated prostate biopsies. Results: Of the 283 patients, 138 (48.8%) had PCa and in 145 (51.2%) a benign prostate disease was diagnosed. PSA, PSAD, and PSADTZ were significantly related to biopsy, and equally able to predict higher pathological stage. The receiver operating curve (AUC) for predicting the presence of PCa in all patients was 58.06 for PSA, 72.13 for PSAD and 78.28 for PSADTZ. In addition, the AUC for predicting higher pathological stage in PCa patients was 65.71 for PSA, 65.46 for PSAD and 69.81 for PSADTZ. For 228 MRI-positive patients, the AUC for predicting the presence of PCa was 61.31 for PSA, 74.00 for PSAD and 80.13 for PSADTZ. No difference among the PSA, PSAD, and PSADTZ was found in 55 MRI-negative patients. Conclusion: The determination of PSADTZ had higher diagnostic accuracy for PCa than that based on PSA or PSAD. For MRI-positive patients, PSADTZ promote a more effective and simple method for PCa detection, and may be useful for decreasing the burden of surveillance prostate biopsies.

ObjectivesThis research aimed to develop a simple and effective acute coronary syndrome (ACS) screening model in order to intervene early and focus on prevention in patients presenting with arteriosclerosis.DesignA case–control study.SettingThe study used a cross-sectional survey to collect data from 2243 patients who completed anonymous electronic medical record (EMR) data and coronary angiography was gathered at a hospital in Shandong Province between December 2013 and April 2016.ParticipantsAdults 18 years old and above diagnosed as ACS or non-ACS according to the records in hospital EMR database, and with completed basic information (age and sex).Predictors54 laboratory biomarkers and demographic factors (age and sex).Statistical analysisA dataset without missing data of all patients' laboratory indicators and demographic factors was divided into training set and validation set after being balanced. After the training set balanced, area under the curve of random forest (AUCRF) and least absolute shrinkage and selection operator (LASSO) regression were used for feature extraction. Then two set random forest models were established with the different feature sets, and the process of comparison and analysis was made to evaluate models for the optimal model including sensitivity, accuracy and AUC receiver operating characteristic curves with the internal validation set.Main outcome measuresTo establish an ACS screening model.ResultsAn RF model with 31 features selected by LASSO with an AUC of 0.616 (95% CI 0.650 to 0.772), a sensitivity of 0.832 and an accuracy of 0.714 in the validation set. The other RF model with 27 features selected by AUCRF with an AUC of 0.621 (95% CI 0.664 to 0.785), a sensitivity of 0.849 and an accuracy of 0.728 in the validation set.ConclusionsThe established ACS screening model with 27 clinical features provides a better performance for practical solution in predicting ACS.

Tea is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, p-coumaroyl-CoA, which is formed from p-coumaric acid catalyzed by p-coumaric acid: CoA ligase (4CL). Herein, we reported two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which were a member of class I and II, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, light and stress-inducible (LSI), and meristem-specific elements, while that of Cs4CL2 had AC and LSI elements only. Moreover, the promoter of Cs4CL1 had two more stress-inducible elements than Cs4CL2 had and the two promoters had six different light-inducible elements. These features suggested their differences in their responses to environmental conditions. Three stress treatments indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assay showed that both recombinant Cs4CL1 and Cs4CL2 transformed p-coumaric acid, ferulic acid and caffeic acid to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze caffeic acid, while the recombinant Cs4CL2 favored to catalyze p-coumaric acid. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

Background This Mendelian randomization study aims to investigate causal associations between genetically predicted insomnia and 14 cardiovascular diseases (CVDs) as well as the potential mediator role of 17 cardiometabolic risk factors. Methods and Results Using genetic association estimates from large genome‐wide association studies and UK Biobank, we performed a 2‐sample Mendelian randomization analysis to estimate the associations of insomnia with 14 CVD conditions in the primary analysis. Then mediation analysis was conducted to explore the potential mediator role of 17 cardiometabolic risk factors using a network Mendelian randomization design. After correcting for multiple testing, genetically predicted insomnia was consistent significantly positively associated with 9 of 14 CVDs, those odds ratios ranged from 1.13 (95% CI, 1.08–1.18) for atrial fibrillation to 1.24 (95% CI, 1.16–1.32) for heart failure. Moreover, genetically predicted insomnia was consistently associated with higher body mass index, triglycerides, and lower high‐density lipoprotein cholesterol, each of which may act as a mediator in the causal pathway from insomnia to several CVD outcomes. Additionally, we found very little evidence to support a causal link between insomnia with abdominal aortic aneurysm, thoracic aortic aneurysm, total cholesterol, low‐density lipoprotein cholesterol, glycemic traits, renal function, and heart rate increase during exercise. Finally, we found no evidence of causal associations of genetically predicted body mass index, high‐density lipoprotein cholesterol, or triglycerides on insomnia. Conclusions This study provides evidence that insomnia is associated with 9 of 14 CVD outcomes, some of which may be partially mediated by 1 or more of higher body mass index, triglycerides, and lower high‐density lipoprotein cholesterol.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC–qTOF–MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Transcription factors (TFs) andWe selected two Arabidopsis TF types and two tobacco CRE types to design aG-box and MYB recognition elements (MREs) were identified in fourPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activatedG-boxes, MREs, and two TF types are appropriate molecular tools for a

IntroductionTranscription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants.ObjectivesWe selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering.MethodsG-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism.ResultsPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P+T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P+T and PAP1 genotypes.ConclusionG-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Complex diseases are believed to be the consequence of intracellular network(s) involving a range of factors. An improved understanding of a disease-predisposing biological network could lead to better identification of genes and pathways that confer disease risk and therefore inform drug development. The group difference in biological networks, as is often characterized by graphs of nodes and edges, is attributable to effects of these nodes and edges. Here we introduced pointwise mutual information (PMI) as a measure of the connection between a pair of nodes with either a linear relationship or nonlinear dependence. We then proposed a PMI-based network regression (PMINR) model to differentiate patterns of network changes (in node or edge) linking a disease outcome. Through simulation studies with various sample sizes and inter-node correlation structures, we showed that PMINR can accurately identify these changes with higher power than current methods and be robust to the network topology. Finally, we illustrated, with publicly available data on lung cancer and gene methylation data on aging and Alzheimer’s disease, an evaluation of the practical performance of PMINR. We concluded that PMI is able to capture the generic inter-node correlation pattern in biological networks, and PMINR is a powerful and efficient approach for biological network analysis.

Background Genome-wide association studies (GWAS) have successfully identified genetic susceptible variants for complex diseases. However, the underlying mechanism of such association remains largely unknown. Most disease-associated genetic variants have been shown to reside in noncoding regions, leading to the hypothesis that regulation of gene expression may be the primary biological mechanism. Current methods to characterize gene expression mediating the effect of genetic variant on diseases, often analyzed one gene at a time and ignored the network structure. The impact of genetic variant can propagate to other genes along the links in the network, then to the final disease. There could be multiple pathways from the genetic variant to the final disease, with each having the chain structure since the first node is one specific SNP (Single Nucleotide Polymorphism) variant and the end is disease outcome. One key but inadequately addressed question is how to measure the between-node connection strength and rank the effects of such chain-type pathways, which can provide statistical evidence to give the priority of some pathways for potential drug development in a cost-effective manner. Results We first introduce the maximal correlation coefficient (MCC) to represent the between-node connection, and then integrate MCC with K shortest paths algorithm to rank and identify the potential pathways from genetic variant to disease. The pathway importance score (PIS) was further provided to quantify the importance of each pathway. We termed this method as “MCC-SP”. Various simulations are conducted to illustrate MCC is a better measurement of the between-node connection strength than other quantities including Pearson correlation, Spearman correlation, distance correlation, mutual information, and maximal information coefficient. Finally, we applied MCC-SP to analyze one real dataset from the Religious Orders Study and the Memory and Aging Project, and successfully detected 2 typical pathways from APOE genotype to Alzheimer’s disease (AD) through gene expression enriched in Alzheimer’s disease pathway. Conclusions MCC-SP has powerful and robust performance in identifying the pathway(s) from the genetic variant to the disease. The source code of MCC-SP is freely available at GitHub (https://github.com/zhuyuchen95/ADnet).

Background Aneurysmal bone cyst (ABC) secondary to Giant Cell Tumor of bone (GCT) is a rare lesion, of which the incidence is about 0.011 to 0.053 per 100,000 every year. There are only a few previous case reports, and most of them occur in the spine, long bones or flat bones. Case presentation We report one case of a patient who complained of “progressive enlargement of the mass on right-hand fifth finger for 5 years with ulceration for 6 months”. After the imaging examination in our hospital, it was diagnosed as a “huge bone tumor on the proximal phalanx of the right-hand fifth finger”, then wide excision and amputation of the fifth finger were made. The pathological examination diagnosed the mass as aneurysmal bone cyst secondary to giant cell tumor, 13 × 8 × 6 cm3, with no local infiltration observed. No recurrence and metastasis occurred 18 months after the operation, and the patient recovered well. Conclusion In this report, we discuss the etiology, diagnosis, differentiation, and management of Aneurysmal bone Cyst secondary to Giant Cell Tumor of bone, and review previous case studies.

Tea is an important economic crop with a 3.02 Gb genome. It accumulates various bioactive compounds, especially catechins, which are closely associated with tea flavor and quality. Catechins are biosynthesized through the phenylpropanoid and flavonoid pathways, with 12 structural genes being involved in their synthesis. However, we found that in Camellia sinensis the understanding of the basic profile of catechins biosynthesis is still unclear. The gene structure, locus, transcript number, transcriptional variation, and function of multigene families have not yet been clarified. Our previous studies demonstrated that the accumulation of flavonoids in tea is species, tissue, and induction specific, which indicates that gene coexpression patterns may be involved in tea catechins and flavonoids biosynthesis. In this paper, we screened candidate genes of multigene families involved in the phenylpropanoid and flavonoid pathways based on an analysis of genome and transcriptome sequence data. The authenticity of candidate genes was verified by PCR cloning, and their function was validated by reverse genetic methods. In the present study, 36 genes from 12 gene families were identified and were accessed in the NCBI database. During this process, some intron retention events of the CsCHI and CsDFR genes were found. Furthermore, the transcriptome sequencing of various tea tissues and subcellular location assays revealed coexpression and colocalization patterns. The correlation analysis showed that CsCHIc, CsF3'H, and CsANRb expression levels are associated significantly with the concentration of soluble PA as well as the expression levels of CsPALc and CsPALf with the concentration of insoluble PA. This work provides insights into catechins metabolism in tea and provides a foundation for future studies.

BACKGROUND Sevoflurane inhalation induction is widely used in pediatric anesthesia, but the minimum alveolar concentration for endotracheal intubation (MACEI) when combined with neuromuscular blockade in neonates has been largely unexplored. This study assessed the MACEI of sevoflurane combined with cisatracurium in neonates. MATERIAL AND METHODS Anesthesia induction was commenced by inhaling 4% sevoflurane with 2 l/min of 100% oxygen via mask. Neonates were administered cisatracurium 0.2 mg/kg followed by adjustment of inspired sevoflurane to target end-tidal concentration based on intubation condition of the preceding subject. When the steady-state end-tidal sevoflurane concentration target was maintained for at least 15 min, endotracheal intubation by direct laryngoscope was performed. The intubation condition was considered failed if either heart rate (HR) after intubation increased by 20% or mean arterial blood pressure (MAP) by 30% or more than that before intubation. Otherwise, the intubation condition was regarded as successful. Dixon's up-and-down method was used with 0.2% as the step size to determine the target end-tidal sevoflurane concentration. RESULTS The MACEI of sevoflurane combined with cisatracurium in neonates was 2.76±0.24%. Using probit analysis, the 50% effective end-tidal sevoflurane concentration (ED50) for successful condition of endotracheal intubation was 2.61% (95%CI 2.07-2.88%) and the 95% effective end-tidal sevoflurane concentration (ED95) was 3.28% (95%CI 2.95-7.19%). Hypotension and bradycardia occurred in 2 neonates during induction. CONCLUSIONS Sevoflurane combined with cisatracurium is feasible and effective for intubation in neonates, and the MACEI of sevoflurane in this subpopulation is 2.76±0.24%. However, cardiovascular adverse effects should be taken into consideration.

Background Tea plants [Camellia sinensis (L.) O. Kuntze] can produce one of the three most widely popular non-alcoholic beverages throughout the world. Polyphenols and volatiles are the main functional ingredients determining tea’s quality and flavor; however, the biotic or abiotic factors affecting tea polyphenol biosynthesis are unclear. This paper focuses on the molecular mechanisms of sucrose on polyphenol biosynthesis and volatile composition variation in tea plants. Results Metabolic analysis showed that the total content of anthocyanins, catechins, and proanthocyanidins(PAs) increased with sucrose, and they accumulated most significantly after 14 days of treatment. Transcriptomic analysis revealed 8384 and 5571 differentially expressed genes in 2-day and 14-day sucrose-treated tea plants compared with control-treated plants. Most of the structural genes and transcription factors (TFs) involved in polyphenol biosynthesis were significantly up-regulated after 2d. Among these transcripts, the predicted genes encoding glutathione S-transferase (GST), ATP-binding cassette transporters (ABC transporters), and multidrug and toxic compound extrusion transporters (MATE transporters) appeared up regulated. Correspondingly, ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) analysis revealed that the content of non-galloylated catechins and oligomeric PAs decreased in the upper-stem and increased in the lower-stem significantly, especially catechin (C), epicatechin (EC), and their oligomeric PAs. This result suggests that the related flavonoids were transported downward in the stem by transporters. GC/MS data implied that four types of volatile compounds, namely terpene derivatives, aromatic derivatives, lipid derivatives, and others, were accumulated differently after in vitro sucrose treatment. Conclusions Our data demonstrated that sucrose regulates polyphenol biosynthesis in Camellia sinensis by altering the expression of transcription factor genes and pathway genes. Additionally, sucrose promotes the transport of polyphenols and changes the aroma composition in tea plant. Electronic supplementary material The online version of this article (10.1186/s12870-018-1335-0) contains supplementary material, which is available to authorized users.

Table S1. Effects of sucrose on volatile compounds in leaves of tea plants using GC/ MS. Note: The data represents the mean value of three biological replications. The red indicates significant up-regulation; green indicates significant down-regulation; blue indicates no difference;. Digit indicates the ratio of Suc / Control. (DOCX 40 kb)

Table S4. Statistics of assembly quality. Note: Total Consensus Sequences represents the all assembled Unigenes, Distinct Clusters represents the cluster Unigenes; the same cluster contains some highly similar (more than 70%) Unigenes and these may come from same gene or homologous gene, Distinct Singletons represents Unigenes from a single gene. (DOCX 21 kb)

Table S3. Statistics of sequencing output. Note: Q20 percentage is the proportion of nucleotides with quality value larger than 20, N percentage is proportion of unknown nucleotides in clean reads, GC percentage is proportion of guanidine and cytosine nucleotides among total nucleotides. (DOCX 20 kb)

Table S2. Effects of sucrose on the expression of genes related to aroma. (DOCX 26 kb)

Tea is one of the most widely consumed nonalcoholic beverages worldwide. Polyphenols are nutritional compounds present in the leaves of tea plants. Although numerous genes are functionally characterized to encode enzymes that catalyze the formation of diverse polyphenolic metabolites, transcriptional regulation of those different pathways such as late steps of the proanthcoyanidin (PA) pathway remains unclear. In this study, using different tea transcriptome databases, we screened at least 140 R2R3-MYB transcription factors (TFs) and grouped them according to the basic function domains of the R2R3 MYB TF superfamily. Among 140 R2R3 TFs, CsMYB5a and CsMYB5e were chosen for analysis because they may be involved in PA biosynthesis regulation. CsMYB5a-overexpressing tobacco plants exhibited downregulated anthocyanin accumulation but a high polymeric PA content in the flowers. Overexpression of CsMYB5e in tobacco plants did not change the anthocyanin content but increased the dimethylaminocinnamaldehyde-stained PA content. RNA-seq and qRT-PCR analyses revealed that genes related to PA and anthocyanin biosynthesis pathways were markedly upregulated in both CsMYB5a- and CsMYB5e-overexpressing flowers. Three UGTs and four GSTs were identified as involved in PA and anthocyanin glycosylation and transportation in transgenic plants. These results provide new insights into the regulation of PA and anthocyanin biosynthesis in Camellia sinensis.

Green tea (Camellia sinensis, Cs) abundantly produces a diverse array of phenylpropanoid compounds benefiting human health. To date, the regulation of the phenylpropanoid biosynthesis in tea remains to be investigated. Here, we report a cDNA isolated from leaf tissues, which encodes a R2R3-MYB transcription factor. Amino acid sequence alignment and phylogenetic analysis indicate that it is a member of the MYB4-subgroup and named as CsMYB4a. Transcriptional and metabolic analyses show that the expression profile of CsMYB4a is negatively correlated to the accumulation of six flavan-3-ols and other phenolic acids. GFP fusion analysis shows CsMYB4a’s localization in the nucleus. Promoters of five tea phenylpropanoid pathway genes are isolated and characterized to contain four types of AC-elements, which are targets of MYB4 members. Interaction of CsMYB4a and five promoters shows that CsMYB4a decreases all five promoters’ activity. To further characterize its function, CsMYB4a is overexpressed in tobacco plants. The resulting transgenic plants show dwarf, shrinking and yellowish leaf, and early senescence phenotypes. A further genome-wide transcriptomic analysis reveals that the expression levels of 20 tobacco genes involved in the shikimate and the phenylpropanoid pathways are significantly downregulated in transgenic tobacco plants. UPLC-MS and HPLC based metabolic profiling reveals significant reduction of total lignin content, rutin, chlorogenic acid, and phenylalanine in CsMYB4a transgenic tobacco plants. Promoter sequence analysis of the 20 tobacco genes characterizes four types of AC-elements. Further CsMYB4a-AC element and CsMYB4a-promoter interaction analyses indicate that the negative regulation of CsMYB4a on the shikimate and phenylpropanoid pathways in tobacco is via reducing promoter activity. Taken together, all data indicate that CsMYB4a negatively regulates the phenylpropanoid and shikimate pathways. Highlight: A tea (Camellia sinensis) MYB4a is characterized to encode a R2R3-MYB transcription factor. It is shown to repressively control the phenylpropanoid and shikimate pathway.