Bimetallic Ru/Ru-Catalyzed Asymmetric One-Pot Sequential Hydrogenations for the Stereodivergent Synthesis of Chiral Lactones.

Asymmetric sequential hydrogenations of α-methylene γ- or δ-keto carboxylic acids are established in one-pot using a bimetallic Ru/Ru catalyst system, achieving the stereodivergent synthesis of all four stereoisomers of both chiral γ- and δ-lactones with two non-vicinal carbon stereocenters in high yields (up to 99%) and with excellent stereoselectivities (up to >99% ee and >20:1 dr). The compatibility of the two chiral Ru catalyst systems is investigated in detail, and it is found that the basicity of the reaction system plays a key role in the sequential hydrogenation processes. The protocol can be performed on a gram-scale with a low catalyst loading (up to 11000 S/C) and the resulting products allow for many transformations, particularly for the synthesis of several key intermediates useful for the preparation of chiral drugs and natural products.

Value of T lymphocyte subset detection in cervical intraepithelial neoplasia.

OBJECTIVE: This study aimed to explore the value of T lymphocyte subset detection in cervical intraepithelial neoplasia (CIN). METHODS: In this retrospective analysis, T lymphocyte subsets in 186 CIN patients were detected. Venous blood T lymphocyte subsets were analyzed in patients with different CIN grades, and Spearman correlation analysis was conducted between CIN grade and T lymphocyte subsets. RESULTS: (1) There were significant differences in the CD3+, CD4+, CD8+, and CD4+/CD8+ levels before and 1, 2, and 3 months after treatment (P<0.05). Furthermore, significant differences were found in CD3+, CD4+, CD8+, and CD4+/CD8+ between every pair of time points (P<0.05). (2) Comparison of human papillomavirus distribution in patients with different CIN grades showed P<0.05. (3) The level of T lymphocyte subsets in the venous blood of patients with different CIN grades was compared, and significant differences were found, P<0.05. Higher CIN grade was associated with lower levels of CD3+, CD4+ and CD4+/CD8+, as well as higher level of CD8+. (4) Spearman analysis showed that CIN grade was negatively correlated with the levels of CD3+, CD4+, and CD4+/CD8+ (P<0.05) and positively correlated with the level of CD8+ (P<0.05). CONCLUSION: The levels of T lymphocyte subsets were found to be closely associated with the severity of CIN. Therefore, the detection of T lymphocyte subsets in venous blood could be a valuable clinical tool for predicting the presence and degree of CIN.

Quantitative proteomics revealed the transition of ergosterol biosynthesis and drug transporters processes during the development of fungal fluconazole resistance.

Fungal infections and antifungal resistance are the increasing global public health concerns. Mechanisms of fungal resistance include alterations in drug-target interactions, detoxification by high expression of drug efflux transporters, and permeability barriers associated with biofilms. However, the systematic panorama and dynamic changes of the relevant biological processes of fungal drug resistance acquisition remain limited. In this study, we developed a yeast model of resistance to prolonged fluconazole treatment and utilized the isobaric labels TMT (tandem mass tag)-based quantitative proteomics to analyze the proteome composition and changes in native, short-time fluconazole stimulated and drug-resistant strains. The proteome exhibited significant dynamic range at the beginning of treatment but returned to normal condition upon acquisition drug resistance. The sterol pathway responded strongly under a short time of fluconazole treatment, with increased transcript levels of most enzymes facilitating greater protein expression. With the drug resistance acquisition, the sterol pathway returned to normal state, while the expression of efflux pump proteins increased obviously on the transcription level. Finally, multiple efflux pump proteins showed high expression in drug-resistant strain. Thus, families of sterol pathway and efflux pump proteins, which are closely associated with drug resistance mechanisms, may play different roles at different nodes in the process of drug resistance acquisition. Our findings uncover the relatively important role of efflux pump proteins in the acquisition of fluconazole resistance and highlight its potential as the vital antifungal targets.

[Quantitative proteomics reveal the potential biological functions of the deubiquitinating enzyme Ubp14 in Saccharomyces cerevisiae].

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.

Data Visualization and Interaction of Urban Traffic Logistics Management System Using WebGIS.

At present, the large amount of data generated by transportation and logistics in cities brings great difficulties to data management and operation. The purpose is to explore the applicability of WebGIS and expand the application of intelligent interactive urban traffic logistics management. An urban traffic logistics management system is designed for urban traffic route assignment and intelligent interaction based on the WebGIS system and the basic principles and related algorithms of urban traffic route assignment. Then, the operation of the system under different shortest path algorithms and different path assignment algorithms is discussed. As a result, the Dijkstra algorithm runs faster than the Floyd algorithm. The comparison of three different road assignment algorithms shows that the no road assignment algorithm has the smallest average time consumption, which is 264.43 ms. The running time of the continuous average algorithm is the same as that of the user balance algorithm. The WebGIS system has practical application value compared with the TransCAD system and Cube system. This article proposes that the interactive function of the system can be used normally. The vehicle information of the test sample points can be displayed, and the function realization effect is good. The research results can provide scientific references for the follow-up research on intelligent transportation.

HBx promotes hepatocarcinogenesis by enhancing phosphorylation and blocking ubiquitinylation of UHRF2.

BACKGROUND AND AIMS: The major cause of Hepatocellular carcinoma (HCC) is acute or chronic infection caused by hepatotropic viruses and HBV infection is the main cause. UHRF2, a ubiquitin-protein ligase E3, is associated with cancer development. This study aimed to investigate the connection and mechanism between UHRF2 and HBV-associated HCC. METHODS: The expression of UHRF2 in human HBV-positive HCC tissues and paracancerous tissues was detected by western blot and tissue microarray. The effects of UHRF2 on invasion, migration and proliferation were detected in HBV-positive hepatoma cell lines. Furthermore, western blot, immunofluorescence, Co-immunoprecipitation and ubiquitination assays were used to explore the relationship and mechanism between UHRF2 and HBV-associated HCC. RESULTS: HBV-positive HCC tissues had higher UHRF2 expression levels than adjacent non-tumor tissues. The HBV-positive HCC patients with a low UHRF2 level in cancer tissues had longer overall and recurrence-free survival compared with those with a high UHRF2 level. UHRF2 induced invasion, migration and proliferation in human HBV-positive HCC cell lines HepG2.2.15 and Hep AD38(-). HBx, an encoding protein of HBV, maintained the stability of UHRF2 by blocking the ubiquitination of UHRF2. HBx up-regulated CDK2 expression through ETS1. UHRF2 bound to CDK2 directly and enhanced UHRF2 phosphorylation at serine 643. CONCLUSIONS: These results suggest that HBx-ETS1-CDK2-UHRF2 pathway plays an important role in the pathogenesis of HBV-associated HCC and represents new therapeutic targets for human HCC. CLINICAL TRIALS REGISTRATION: ChiCTR2000041416.

Deubiquitinase Ubp3 enhances the proteasomal degradation of key enzymes in sterol homeostasis.

Sterol homeostasis is tightly controlled by molecules that are highly conserved from yeast to humans, the dysregulation of which plays critical roles in the development of antifungal resistance and various cardiovascular diseases. Previous studies have shown that sterol homeostasis is regulated by the ubiquitin-proteasome system. Two E3 ubiquitin ligases, Hrd1 and Doa10, are known to mediate the proteasomal degradation of 3-hydroxy-3-methylglutaryl-CoA reductase Hmg2 and squalene epoxidase Erg1 with accumulation of the toxic sterols in cells, but the deubiquitinases (DUBs) involved are unclear. Here, we screened for DUBs responsible for sterol homeostasis using yeast strains from a DUB-deletion library. The defective growth observed in ubp3-deleted (ubp3Δ) yeast upon fluconazole treatment suggests that lack of Ubp3 disrupts sterol homeostasis. Deep-coverage quantitative proteomics reveals that ergosterol biosynthesis is rerouted into a sterol pathway that generates toxic products in the absence of Ubp3. Further genetic and biochemical analysis indicated that Ubp3 enhances the proteasome's ability to degrade the ergosterol biosynthetic enzymes Erg1 and Erg3. The retardation of ergosterol enzyme degradation in the ubp3Δ strain resulted in the severe accumulation of the intermediate lanosterol and a branched toxic sterol, and ultimately disrupted sterol homeostasis and led to the fluconazole susceptibility. Our findings uncover a role for Ubp3 in sterol homeostasis and highlight its potential as a new antifungal target.

Over-Expression of LcPDS, LcZDS, and LcCRTISO, Genes From Wolfberry for Carotenoid Biosynthesis, Enhanced Carotenoid Accumulation, and Salt Tolerance in Tobacco.

It is of great importance to combine stress tolerance and plant quality for breeding research. In this study, the role of phytoene desaturase (PDS), ζ-carotene desaturase (ZDS) and carotene isomerase (CRTISO) in the carotenoid biosynthesis are correlated and compared. The three genes were derived from Lycium chinenses and involved in the desaturation of tetraterpene. Their over-expression significantly increased carotenoid accumulation and enhanced photosynthesis and salt tolerance in transgenic tobacco. Up-regulation of almost all the genes involved in the carotenoid biosynthesis pathway and only significant down-regulation of lycopene ε-cyclase (ε-LCY) gene were detected in those transgenic plants. Under salt stress, proline content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were significantly increased, whereas malonaldehyde (MDA) and hydrogen peroxide (H2O2) accumulated less in the transgenic plants. The genes encoding ascorbate peroxidase (APX), CAT, POD, SOD, and pyrroline-5-carboxylate reductase (P5CR) were shown to responsive up-regulated significantly under the salt stress in the transgenic plants. This study indicated that LcPDS, LcZDS, and LcCRTISO have the potential to improve carotenoid content and salt tolerance in higher plant breeding.

Urine proteome of COVID-19 patients.

The atypical pneumonia (COVID-19) caused by SARS-CoV-2 is a serious threat to global public health. However, early detection and effective prediction of patients with mild to severe symptoms remain challenging. The proteomic profiling of urine samples from healthy individuals, mild and severe COVID-19 positive patients with comorbidities can be clearly differentiated. Multiple pathways have been compromised after the COVID-19 infection, including the dysregulation of complement activation, platelet degranulation, lipoprotein metabolic process and response to hypoxia. This study demonstrates the COVID-19 pathophysiology related molecular alterations could be detected in the urine and the potential application in auxiliary diagnosis of COVID-19.

Overexpression of lycopene ε-cyclase gene from lycium chinense confers tolerance to chilling stress in Arabidopsis thaliana.

Lutein plays an important role in protecting the photosynthetic apparatus from photodamage and eliminating ROS to render normal physiological function of cells. As a rate-limiting step for lutein synthesis in plants, lycopene ε-cyclase catalyzes lycopene to δ-carotene. We cloned a lycopene ε-cyclase gene (Lcε-LYC) from Lycium chinense (L. chinense), a deciduous woody perennial halophyte growing in various environmental conditions. The Lcε-LYC gene has an ORF of 1569bp encoding a protein of 522 aa. The deduced amino acid sequence of Lcε-LYC gene has higher homology with LycEs in other plants, such as Nicotiana tabacum and Solanum tuberosum. When L. chinense was exposed to chilling stress, relative expression of Lcε-LYC increased. To study the protective role of Lcε-LYC against chilling stress, we overexpressed the Lcε-LYC gene in Arabidopsis thaliana. Lcε-LYC overexpression led to an increase of lutein accumulation in transgenic A. thaliana, and the content of lutein decreased when transgenics were under cold conditions. In addition, the transgenic plants under chilling stress displayed higher activities of superoxide dismutase (SOD) and peroxidase (POD) and less H2O2 and malondialdehyde (MDA) than the control. Moreover, the photosynthesis rate, photosystem II activity (Fv/fm), and Non-photochemical quenching (NPQ) also increased in the transgenetic plants. On the whole, overexpression of Lcε-LYC ameliorates photoinhibition and photooxidation, and decreases the sensitivity of photosynthesis to chilling stress in transgenic plants.

De novo characterization of the Lycium chinense Mill. leaf transcriptome and analysis of candidate genes involved in carotenoid biosynthesis.

Lycium chinense Mill. (Chinese wolfberry), enriching in carotenoids, is an important Chinese herbal medicine. However, studies on the functional genomics research, especially the carotenoid biosynthesis and accumulation, are limited because of insufficiently available datasets. RNA-Seq was performed by the Illumina sequencing platform. Approximately 26 million clean reads were generated after filtering. Clean reads were assembled by SOAPdenovo and subsequently annotated. Among all 61,595 unigenes, 37,816 (61.39%), 25,266 (41.02%), and 17,598 (28.57%) unigenes were annotated in NCBI non-redundant protein, Swiss-Prot, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. A total of 16,073 and 11,394 unigenes were assigned to Gene Ontology and Cluster of Orthologous Group, respectively. Furthermore, the majority of genes encoding the enzymes in the carotenoid biosynthesis pathway were identified in the unigene datasets. We first found several genes related to L. chinense carotenoid biosynthesis. The expression levels and the biological functions of these genes involved in carotenoid biosynthesis in the leaf and the green ripening fruit were further confirmed by qPCR and high performance liquid chromatography (HPLC). In the present study, we first characterized the transcriptome of L. chinense leaf, which may provide useful data for functional genomics investigations in L. chinense in the future. And essential genes involved in the carotenoid biosynthesis pathway may contribute to elucidate the expression patterns in different stages of development and fruit ripening and the specific mechanisms of carotenoid biosynthesis/accumulation in L. chinense.

Cloning and characterization of a novel ß-carotene hydroxylase gene from Lycium barbarum and its expression in Escherichia coli.

Lycium barbarum contains high levels of zeaxanthin, which is produced by the conversion of ß-carotene into zeaxanthin. ß-Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding ß-carotene hydroxylase (Chyb) from the L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3'-rapid amplification of cDNA end assay encoding a deduced Chyb protein (34.8 kDa) with a theoretical isoelectric point of 8.36. A bioinformatics analysis showed that the L. barbarum Chyb was located in the chloroplast. Further, to investigate the catalytic activity of the L. barbarum Chyb, a complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that Chyb can catalyze ß-carotene to produce zeaxanthin. Thus, this study suggests that L. barbarum ß-carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in E. coli.

The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli.

L-cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of L-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in L-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in L-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to L-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for L-cysteine tolerance in E. coli cells. However, L-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent L-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of L-cysteine, the transformants exhibited more L-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in L: -cysteine tolerance probably due to its export ability and that TolC overexpression is effective for L-cysteine production in E. coli.