Transcriptomic and targeted metabolomic analyses provide insights into the flavonoids biosynthesis in the flowers of Lonicera macranthoides.

BACKGROUND: Flavonoids are one of the bioactive ingredients of Lonicera macranthoides (L. macranthoides), however, their biosynthesis in the flower is still unclear. In this study, combined transcriptomic and targeted metabolomic analyses were performed to clarify the flavonoids biosynthesis during flowering of L. macranthoides. RESULTS: In the three sample groups, GB_vs_WB, GB_vs_WF and GB_vs_GF, there were 25, 22 and 18 differentially expressed genes (DEGs) in flavonoids biosynthetic pathway respectively. A total of 339 flavonoids were detected and quantified at four developmental stages of flower in L. macranthoides. In the three sample groups, 113, 155 and 163 differentially accumulated flavonoids (DAFs) were detected respectively. Among the DAFs, most apigenin derivatives in flavones and most kaempferol derivatives in flavonols were up-regulated. Correlation analysis between DEGs and DAFs showed that the down-regulated expressions of the CHS, DFR, C4H, F3'H, CCoAOMT_32 and the up-regulated expressions of the two HCTs resulted in down-regulated levels of dihydroquercetin, epigallocatechin and up-regulated level of kaempferol-3-O-(6''-O-acetyl)-glucoside, cosmosiin and apigenin-4'-O-glucoside. The down-regulated expressions of F3H and FLS decreased the contents of 7 metabolites, including naringenin chalcone, proanthocyanidin B2, B3, B4, C1, limocitrin-3,7-di-O-glucoside and limocitrin-3-O-sophoroside. CONCLUSION: The findings are helpful for genetic improvement of varieties in L.macranthoides.

Optimization of mammalian expression vector by cis-regulatory element combinations.

The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot. The receptor binding domain (RBD) sequence from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein was used to replace the eGFP sequence in the expression vector and the RBD expression was detected by qRT-PCR and western blot. The results showed that protein expression can be regulated by optimizing the combination of cis-acting elements. The vector with the CMV enhancer, EF-1α core promoter, and TEF terminator was found to express approximately threefold higher eGFP than the unmodified vector in different animal cells, as well as 2.63-fold higher recombinant RBD protein than the original vector in HEK-293T cells. Moreover, we suggest that combinations of multiple regulatory elements capable of regulating gene expression do not necessarily exhibit synergistic effects to enhance expression further. Overall, our findings provide insights into biological applications that require the regulation of gene expression and will help to optimize expression vectors for biosynthesis and other fields. Additionally, we provide valuable insights into the production of RBD proteins, which may aid in developing reagents for diagnosis and treatment during the COVID-19 pandemic.

Desulfurative Ni-Catalyzed Reductive Cross-Coupling of Benzyl Mercaptans/Mercaptoacetates with Aryl Halides.

The C-S activation and sulfur removal from native thiols is challenging, which limits their application as feedstock materials in organic synthesis despite their natural abundance. Herein, we introduce a per-/polyfluoroaryl moiety, which serves as a redox-active scaffold, into sp3-hybridized thiols to activate the C-S bond. Using a Ni catalyst with MgBr2 as an additive, the S group can be removed to yield an aliphatic radical that can react with an aryl halide in a reductive cross-coupling.

SmFLS negatively regulates peel coloring of eggplant (Solanum melongena) under high temperature.

The SmFLS gene was cloned from eggplant and has an ORF of 1014 bp encoding 337 amino acids. The deduced protein sequence of SmFLS was 88.07% and 84.94% identical to homologs encoded by StFLS in Solanum tuberosum and SlFLS in S. lycopersicum, respectively. SmFLS contains typical DIOX_N and 2OG-Fe (II)_Oxy functional domains, as well as five strictly conserved amino acid residues (H223, D225, H279, R289, and S291) related to FLS enzyme activity. Phylogenetic tree analysis showed that SmFLS had the closest genetic relationship with the FLS genes in potato and tomato. At a high temperature of 35 °C, the expression level of SmFLS was higher than that of the control in the same period, and it reached extremely significant levels on 15DAF and 20DAF, at which the eggplant peel color became lighter accordingly. Upon overexpression of SmFLS in eggplant, the flavonol content of transformed plants was significantly higher than that of untransformed plants, and the peel color was lighter than that of the control. The results indicate that SmFLS negatively regulates eggplant peel coloration under high temperature.

[Functional annotation of rice WRKY transcription factors based on their transcriptional features].

Transcription factors regulate alteration of transcription levels. Recently, huge amount of transcriptomic data are accumulated via the application of high throughput sequencing technology, and it is reasonable to postulate that in-depth analysis of transcription data could be used to enhance gene annotation. In this study, we chose the gene family of rice WRKY transcription factors. Based on literature search, the transcriptional data under different biological processes, including biotic and abiotic stress, development, and nutrient absorption and hormone treatments were analyzed systematically. To the end, we summarize the list of differentially expressed WRKY genes. We also expect that such information will enrich their functional annotation and also provide direct clues for subsequent functional studies.

Pro-aromatic Dihydroquinazolinones - From Multigram Synthesis to Reagents for Gram-scale Metallaphotoredox Reactions.

Dihydroquinazolinone (DHQZ) has recently been harnessed as a ketone-derived pro-aromatic reagent extensively employed in (metalla)photoredox reactions as versatile group transfer agents. In this work, we outline a column chromatography-free protocol for the multigram-scale synthesis of pro-aromatic DHQZs as well as its use in a gram-scale nickel/photoredox dual-catalyzed cross-coupling in single-batch, photoflow, and simultaneous multiple smaller batches. While the single-batch approach leveraged moderate yields, a simple plug-flow photoreactor also exhibited amenable productivity (up to 45 % yield) despite the use of a heterogeneous base. Meanwhile, performing the metallaphotoredox-catalyzed reaction in multiple smaller batches in an improvised photoreactor facilitated high yields of up to 59 % and good reproducibility, implying a convenient alternative in the absence of photoflow setups.

Benign prostatic hyperplasia associated with white matter hyperintensities in men.

PURPOSE: Benign prostatic hyperplasia (BPH) describes common noncancerous prostate enlargement. BPH is usually associated with lower urinary tract symptoms and an increased risk of cerebrovascular diseases, such as stroke and its recurrence. White matter hyperintensities (WMHs), markers of cerebral injury, increase the risk of stroke, cognitive impairment, dementia, and death. The relationship between BPH and WMHs remains unclear. This study aimed to determine the association between BPH and WMHs. METHODS: A total of 788 male patients from the First Affiliated Hospital of Kunming Medical University from July 2019 to September 2021 were enrolled in this cross-sectional study. BPH was assessed by abdominal ultrasound, and three independent neuroradiologists rated the presence or absence of WMHs. Multiple imputations of chained equations were used to handle missing data. Logistic regression was used to assess the relationship between BPH and WMHs. RESULTS: Patients with BPH presented an increased risk of WMHs with a crude odds ratio (OR) of 2.76 (95% CI, 2.02-3.79) and an adjusted OR of 1.75 (95% CI, 1.24-2.48) after controlling for potential confounding factors in the multivariate logistic regression. CONCLUSION: We found that BPH was closely associated with WMHs in male Chinese individuals.

Pseudorabies Virus Inhibits Expression of Liver X Receptors to Assist Viral Infection.

Pseudorabies virus (PRV) is a contagious herpesvirus that causes Aujeszky's disease and economic losses worldwide. Liver X receptors (LXRs) belong to the nuclear receptor superfamily and are critical for the control of lipid homeostasis. However, the role of LXR in PRV infection has not been fully established. In this study, we found that PRV infection downregulated the mRNA and protein levels of LXRα and LXRß in vitro and in vivo. Furthermore, we discovered that LXR activation suppressed PRV proliferation, while LXR inhibition promoted PRV proliferation. We demonstrated that LXR activation-mediated reduction of cellular cholesterol was critical for the dynamics of PRV entry-dependent clathrin-coated pits. Replenishment of cholesterol restored the dynamics of clathrin-coated pits and PRV entry under LXR activation conditions. Interestingly, T0901317, an LXR agonist, prevented PRV infection in mice. Our results support a model that PRV modulates LXR-regulated cholesterol metabolism to facilitate viral proliferation.

Aromatization as an Impetus to Harness Ketones for Metallaphotoredox-Catalyzed Benzoylation/Benzylation of (Hetero)arenes.

Herein we report ketones as feedstock materials in radical cross-coupling reactions under Ni/photoredox dual catalysis. In this approach, simple condensation first converts ketones into prearomatic intermediates that then act as activated radical sources for cross-coupling with aryl halides. Our strategy enables the direct benzylation/benzoylation of (hetero)arenes under mild reaction conditions with high functional group tolerance.

[An epidemiological study of chronic obstructive pulmonary disease in greenhouse farmers in Liaoning Province from 2006 to 2009].

OBJECTIVE: To investigate the prevalence of chronic obstructive pulmonary disease (COPD) in greenhouse farmers in Liaoning Province from 2006 to 2009. METHODS: By stratified-cluster random sampling, a survey of 5420 greenhouse farmers working in 835 plastic greenhouses was performed using uniform questionnaires in Shenyang, Chaoyang, and Jinzhou during 2006 and 2009. Physical examination and lung function tests were performed for all of the farmer. Blood routine examinations, blood gas analysis, and chest X-ray were performed for some of the subjects with respiratory symptoms and healthy controls. The chi-square test was used for comparison of the 2 sample rates, and the chi-square segmentation method for more. RESULTS: The overall prevalence of COPD in greenhouse farmers in Liaoning Province was 17.5% (947/5420). Most patients were elderly, especially at the age of 50 years or older. The prevalence of COPD in greenhouse farmers in the plain areas was 15.6% (464/2981), which was lower than that in the mountains (20.2%, 246/1220) and coastal areas (19.4%, 237/1219), with a significant difference (the value of χ(2) were 13.04 and 9.35, all P < 0.0125). The prevalence of COPD in vegetable greenhouse farmers was 12.6% (273/2168), which was lower than that in fungus greenhouse farmers (24.3%, 263/1084), poultry greenhouse farmers (20.8%, 169/813) and flowers greenhouse farmers (17.9%, 242/1355), with a significant difference (the value of χ(2) were 71.49, 31.44 and 18.54, all P < 0.007). The prevalence of COPD was the highest (28.3%, 137/485) in farmers who had worked in the greenhouses for 3 to 5 years. CONCLUSIONS: The overall prevalence of COPD in greenhouse farmers in Liaoning Province was higher than that reported in the rural areas nationally. Attention should be paid to the prevention and control of COPD in greenshouse farmers.

[Replacement of third-generation cephalosporins by piperacillin-tazobactam decreases colonization of extended-spectrum beta-lactamases-producing intestinal Escherichia coli].

OBJECTIVE: To investigate the role of replacement of third-generation cephalosporins by piperacillin-tazobactam (pip-tazo) in influencing the colonization of extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli (E. coli) in intestinal tract. METHODS: The study was divided into two phases lasting altogether 9 months, namely the pre-replacement phase (phase I, 3 months) and replacement phase (phase II, 6 months). In the latter phase, third-generation cephalosporins was restricted and replaced by pip-tazo. In phase I and phase IIb (the last 3 months of phase II), clinical data and rectal swab were taken for E. coli isolation as follow: within 24 hrs after admission (baseline screening), every week and 48 hrs before discharge. ESBLs production was detected with double disc test. Acquisition rate of ESBLs-producing E. coli were calculated both in ES1 group (patients' rectal swab collected and tests at least 2 times) and ES2 group (ES1 but with negative ESBLs either at the time of screening on admission or at anytime during the hospital stay). Continuous variable was compared using unpaired t-test and categorical variables was compared using Pearson Chi square test. Fisher's exact test was used in the two phases. RESULTS: In phase IIb, as compared with in phase I, the total consumption of antibiotics other than pip-tazo was reduced by 38.40%, the third-generation cephalosporins consumption was reduced by 70.11%, but pip-tazo consumption was raised by 895.35%. Meanwhile, the acquisition rate of ESBLs-producing E. coli in rectal swab was significantly decreased in phase IIb as compared with phase I (11.4% vs 24.0%) in ES1 group and the same is true in ES2 group (11.8% vs 27.9%). CONCLUSION: Replacement of third-generation cephalosporin with pip-tazo can reduce colonization of ESBLs--producing E. coli in intestinal tract.

[Expression and autophosphorylation analysis of XA21 and PI-D2 protein kinases in Saccharomyces cerevisiae].

Rice bacterial blight and blast are the most crucial rice disease. Xa21 confers resistance to bacterial blight, while Pi-d2 confers resistance to rice blast. Both Xa21 and Pi-d2 encode receptor kinase-like proteins. Biochemical properties of XA21 kinase expressed in bacterial were characterized in our previous report. In this study, both XA21 and PI-D2 kinase domain were PCR amplified and cloned into yeast expression vector pEGH via recombinational cloning strategy, kinase proteins expressed in eukaryotic yeast system was purified and autophosphorylation assay was carried out. The results indicated that XA21 and PI-D2 protein can be detected by SDS-PAGE and showed expected molecular weight. Autophosphorylation assay indicated that yeast expressed XA21 and PI-D2 were active when incubated with P32 labelled ATP. The experiment provided basic materials for biochemical prosperity analysis, protein-protein interaction and substrate screening research.

[Advances of rice bacterial blight disease resistance gene Xa21].

Gene Xa21 is the first disease resistance gene cloned from rice (Oryza sativa L.), which encodes a receptor-like kinase and confers broad spectrum resistance against Xanthomonas oryzae pv. oryzae (Xoo). In this paper, the discovery, mapping and cloning of Xa21 were reviewed briefly. The molecular mechanism of resistance and its breeding application, including biochemical properties of the kinase domain of XA21, AvrXa21 identification, the disease resistance pathway mediated by Xa21, and the mechanism of disease resistance were summarized. Future perspective on Xa21-related research is also discussed.

Synthesis and evaluation of gadolinium complexes based on PAMAM as MRI contrast agents.

Diethylenetriaminepentaacetic acid (DTPA) and pyridoxamine (PM) were incorporated into the amine groups on the surface of ammonia-core poly(amidoamine) dendrimers (PAMAM, Generation 2.0-5.0) to obtain dendritic ligands. These dendritic ligands were reacted with gadolinium chloride to yield the corresponding dendritic gadolinium (Gd) complexes. The dendritic ligands and their gadolinium complexes were characterized by(1)HNMR, IR, UV and elemental analysis. Relaxivity studies showed that the dendritic gadolinium complexes possessed higher relaxation effectiveness compared with the clinically used Gd-DTPA. After administration of the dendritic gadolinium complexes (0.09 mmol kg(-1) ) to rats, magnetic resonance imaging of the liver indicated that the dendritic gadolinium complexes containing pyridoxamine groups enhanced the contrast of the MR images of the liver, provided prolonged intravascular duration and produced highly contrasted visualization of blood vessels.

Association between the ABCA1-565C/T gene promoter polymorphism and coronary heart disease severity and cholesterol efflux in the Chinese Han population.

BACKGROUND: ABCA1 -565C/T gene promoter variants have been associated with the severity of coronary artery disease in Western populations. The purpose of our study was to investigate the association between the -565C/T gene polymorphism and coronary artery disease severity and cholesterol efflux in the Chinese Han population. METHODS: A cohort of 298 acute coronary syndrome (ACS) patients and 541 healthy controls was genotyped using the highly sensitive ligase detection reaction. ABCA1 -565C/T genotype was correlated with the clinical features of 164 acute myocardial infarction (AMI) patients. Monocytes from patients with various -565C/T gene polymorphisms were isolated and differentiated into foam cells by coincubation with [(3)H]-labeled acetyl-low-density lipoprotein cholesterol. ABCA1 mRNA and protein expression levels were evaluated, as well as cellular cholesterol efflux. RESULTS: The frequency of the TT genotype in the -565C/T polymorphism of ACS patients was significantly increased when compared with controls (0.211 vs. 0.162, p<0.05). The TT genotype, but not the CT or CC genotypes, in the -565C/T gene polymorphism correlated with the severity of the coronary lesion observed in AMI patients. Patients with the TT homozygote genotype also exhibited significantly lower cellular cholesterol efflux (TT [6.37%±0.554%]) levels than controls and also had the lowest levels of ABCA1 mRNA and protein expression among the group of variants. In contrast, cholesterol efflux levels in AMI patients with CT [11.35%±3.975%] and CC ([15.32%±6.293%]) genotypes were not significantly different from controls. CONCLUSIONS: Impaired ABCA1-mediated cholesterol efflux in macrophages may be associated with the severity of the coronary lesions in AMI patients with the TT genotype at the -565C/T gene polymorphism.

Molecular epidemiological study of alpha- and beta-thalassemia in Sihui city.

OBJECTIVE: To investigate alpha- and beta-thalassemia (alpha- and beta-thal) gene frequencies and gene mutation spectrum in the population of Sihui City. METHODS: The umbilical cord blood samples from 1 007 neonates and peripheral blood samples from 1 524 apparently healthy adults for pre-marriage health check in Sihui city were collected for molecular epidemiologic study of alpha- and beta-thal respectively. The diagnostic standard for alpha-thal was the presence of Hb Bart's, and that for beta-thal was both the decrease of mean corpuscular volume (MCV<80 fl) and the increase of Hb A(2) level (> or = 3.5%). The samples of identified subjects with positive thal genotypes were further examined with PCR-based DNA analysis for determining the alpha- or beta-globin gene genotype, while those from subjects with positive genotypes but without mutations known to Chinese subjects were subjected to DNA sequence analysis of beta-globin gene. In addition, the alpha-thal alleles, -alpha(3.7) and -alpha(4.2) were examined in all umbilical cord blood samples. RESULTS AND CONCLUSION: Of all the 1 007 umbilical cord blood samples, 110 were identified as from alpha-thal gene carriers, 3 from patients Hb H disease and 1 from patients with hydrops fetalis, which meant an alpha-thal gene frequency of 11.72% (118/1 007). Three types of alpha-gene deletion were identified in this cohort, with the frequency of 53.4% (--SEA)), 34.7% (-alpha(3.7)) and 11.9% (-alpha(4.2)) respectively. By examining the peripheral venous blood samples from the 1,524 healthy adult subjects, 59 subjects were found to be beta-thal gene carriers with a rate of 3.87% (59/1,524), whose genotypes were determined and from whom 7 beta-thal mutations were identified. Of these 59 beta-thal gene carriers, 11 were diagnosed as having heterozygotes compound for beta- and alpha-thal genes with the deletion of the --(SEA) in 7 cases and -alpha(3.7) in 4 cases respectively, showing an incidence of 0.72% (11/1,524). The three commonest point mutations, beta CD41-42 (-CTTT) frameshift mutation, beta IVS2-654(C-->T) aberrant splicing mutation and beta-28 (A-->G) transcription mutation occurred with a total frequency of 84.75% among subjects with beta-thal allele mutations. In addition, a novel mutation, beta-globin gene promoter -90 (C-->T) allele was detected for the first time in Chinese subjects.

[The effects of Erigeron breviscapus preparation on the imaging and neuronal metabolites at the early time points after reperfusion following the ischemic cerebral injury in rats].

OBJECTIVE: To investigate the effects of Erigeron breviscapus preparation on the imaging and neuronal metabolites after reperfusion in the ischemic cerebral injury in rats. METHOD: Twenty-three male Sprague Dawley rats with an average body weight of (165 +/- 15) g (mean +/- S) were used, and were randomly divided into two groups: group A rats (n = 11) underwent an operation of ischemic brain injury, group B rats (n = 12) underwent the same operation and received the treatment of Erigeron breviscapus preparation (1.5 mg.kg-1 weight, i.p.). The right middle cerebral artery occlusion (MCAO) of rats in both groups was induced by 5/0 nylon suture for 2 hours. The reperfusion was conducted for four hours and six hours respectively following MCAO. T2 weighted-imaging (T2WI) and 1H-magnetic resonance spectroscopy (1H-MRS) were performed, to study the changes of the imaging and the neuronal metabolites N-acetyl aspartate (NAA), creatine/phosphocreatine (Cr/PCr), choline (Cho) and lactose (Lac) in cerebrum following cerebral ischemia. RESULT: The hyperintense signals in the right cerebrum in group B decreased compared with those in group A, the T2 values decreased, the level of NAA increased, the ratio of Cr/NAA and Cho/NAA decreased, and no lactose was observed. The brain surface vessels of rats in group B were in the state of dilation. CONCLUSION: Erigeron breviscapus preparation is beneficial to the reestablishment of the blood circulation in the ischemic brain, and to the improvement of the neuronal metabolism and survival.

[Risk factors on chronic obstructive pulmonary disease among greenhouse workers in Liaoning province].

OBJECTIVE: To study the risk factors of chronic obstructive pulmonary disease (COPD) among greenhouse farmers in Liaoning province. METHODS: According to geographical differences in Liaoning province, stratified-cluster-random sampling method was used. A total of 5420 greenhouse farmers working in more than 800 greenhouses were investigated with unified questionnaire, physical examination, blood routine examination, blood gas analysis, chest X-ray and pulmonary function tests. Diagnosis of COPD was made according to the criteria for "chronic obstructive pulmonary disease of diagnosis and treatment guidelines" (2007 revision) by the Chinese Respiratory Disease Society. RESULTS: The total number of farmers under study was 5880, including 5420 respondants (92.18%) with effective data. Risk factors were found on age (χ2=32.530, P=0.000), time working in greenhouses every day (χ2=21.311, P=0.000), frequency of ventilation (χ2=9.791, P=0.007) and the location of the greenhouses (χ2=73.645, P=0.000). When using SPSS 17.0 for the single factor logistic regression analysis, results indicated that smoking (OR=1.976, 95%CI: 1.672 - 2.334), age (OR=4.188, 95%CI: 3.718-4.718), greenhouses for fungus (compared with vegetables, OR=1.302, 95%CI: 1.016-1.669), flowers and plants (compared with vegetables greenhouses, OR=1.503, 95%CI: 1.201-1.880) were the risk factors of COPD among greenhouse farmers while the protective factor was the years of working in the greenhouse (OR=0.684, 95%CI: 0.609-0.767). CONCLUSION: The overall morbidity of COPD among greenhouse farmers in Liaoning province was 17.47% with various risk factors.

A polymorphism in the ABCG1 promoter is functionally associated with coronary artery disease in a Chinese Han population.

OBJECTIVE: In this study, we examine the association of single nucleotide polymorphisms (SNPs) of the human ATP binding cassette transporter G1 (ABCG1) gene with atherosclerotic coronary artery disease (CAD) in a Chinese Han population. METHODS: 1021 patients with CAD and 1013 unaffected control subjects were enrolled. PCR-based ligation detection reaction (PCR-LDR) method was used to genotype four SNPs of ABCG1, three (rs2234714, rs2234715 and rs57137919) in the promoter region and one (rs1044317) in the 3'-untranslated region (UTR). RESULTS: The human ABCG1 -367G>A polymorphism (rs57137919) showed a significantly decreased risk for CAD and myocardial infarction (MI) in a dominant model (adjusted OR = 0.73, p = 0.033 for CAD, and adjusted OR = 0.65, p = 0.014 for MI, respectively). The rs57137919 also showed an association with angiographic severity of CAD (multi-vessel vs. single-vessel CAD, adjusted OR = 0.40, p = 0.005). The findings were further supported by luciferase reporter assay, in which the polymorphism impaired reporter gene expression. The ABCG1 -768G>A polymorphism (rs2234714) showed an association with CAD in a recessive model (adjusted OR = 0.64, p = 0.015), but did not demonstrate a functional influence on reporter gene expression in the luciferase reporter assay. CONCLUSIONS: The SNP rs57137919 in the ABCG1 promoter region is functionally associated with a reduced risk of CAD in a Chinese Han population.