Post-synthetic modification-driven ZIF reconstruction and functionalization for efficient SARS-CoV-2 ECL detection.

The emergence of novel pathogens, as well as their frequent variants, raises the significance of developing superior and versatile sensing materials and techniques. Herein, a post-modified zeolitic imidazolate framework (pm-ZIF) was synthesized by using ZIF-67 as a parent MOF, and zinc(II) meso-tetra (4-carboxyphenyl) porphine (ZnTCPP) as a successive exchange ligand. Due to the preservation of the tetrahedral Co-N4 units from the ZIF precursor and the introduced porphyrin luminophores, this hybrid material pm-ZIF/P(Zn) enables the linear electrochemiluminescence (ECL) signal conversion of the target DNA concentration. An efficient biosensor that can be used to quantitatively detect SARS-CoV-2 was therefore constructed. The linear range of the sensor was 10-12-10-8 M, with a limit of detection (LOD) reaching 158 pM. Compared with the traditional amplification-based methods, the duration time of our method is significantly shortened and the quantitation of the SARS-Cov-2 RdRp gene can be completed within twenty minutes at room temperature.

Novel ß1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides.

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a ß-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant ß1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-ß-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, ß1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by ß1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial ß1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides. KEY POINTS: • A novel ß-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9. • EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis. • Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.

2D Zn-Porphyrin-Based Co(II)-MOF with 2-Methylimidazole Sitting Axially on the Paddle-Wheel Units: An Efficient Electrochemiluminescence Bioassay for SARS-CoV-2.

High electrocatalytic activity with tunable luminescence is crucial for the development of electrochemiluminescence (ECL) luminophores. In this study, a porphyrin-based heterobimetallic 2D metal organic framework (MOF), [(ZnTCPP)Co2(MeIm)] (1), is successfully self-assembled from the zinc(II) tetrakis(4-carboxyphenyl)porphine (ZnTCPP) linker and cobalt(II) ions in the presence of 2-methylimidazole (MeIm) by a facile one-pot reaction in methanol at room temperature. On the basis of the experimental results and the theoretical calculations, the MOF 1 contains paddle-wheel [Co2(-CO2)4] secondary building units (SBUs) axially coordinated by a MeIm ligand, which is very beneficial to the electron transfer between the Co(II) ions and oxygen. Combining the photosensitizers ZnTCPP and the electroactive [Co2(-CO2)4] SBUs, the 2D MOF 1 possesses an excellent ECL performance, and can be used as a novel ECL probe for rapid nonamplified detection of the RdRp gene of SARS-CoV-2 with an extremely low limit of detection (≈30 aM).

Recent developments in germanium containing clusters in intermetallics and nanocrystals.

Multimetallic clusters can be described as building blocks in intermetallics, compounds prepared from all metals and/or semi-metals, and in Zintl phases, a subset of intermetallics containing metals with large differences in electronegativity. In many cases, these intermetallic and Zintl phases provide the first clue for the possibilities of bond formation between metals and semi-metals. Recent advances in multimetallic clusters found in Zintl phases and nanoparticles focusing on Ge with transition metals and semi-metals is presented. Colloidal routes to Ge nanocrystals provide an opportunity for kinetically stabilized Ge-metal and Ge-semi-metal bonding. These routes provide crystalline nanoclusters of Ge, hereafter referred to as nanocrystals, that can be structurally characterized. Compositions of Ge nanocrystals containing transition metals, and the semi-metals, Sb, Bi, and Sn, whose structures have recently been elucidated through EXAFS, will be presented along with potential applications.

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

Ginsenoside Ro Ameliorates High-Fat Diet-Induced Obesity and Insulin Resistance in Mice via Activation of the G Protein-Coupled Bile Acid Receptor 5 Pathway.

Obesity, a well known risk factor in multiple metabolic diseases, is dramatically increasing worldwide. Ginsenosides extracted from ginseng have been reported against obesity and the associated metabolic disorders. As a subtype of ginsenoside, ginsenoside Ro is a critical constituent of ginseng. However, its specific effects on obesity remain unknown. G protein-coupled bile acid receptor 5 (TGR5) (also known as GPBAR1) is a bile acid membrane receptor, widely expressed in human tissues contributing to various metabolic processes to confer the regulations of glucose and lipid homeostasis. TGR5 has displayed potential as a therapeutic target for the treatment of metabolic disorders. Here, we explore the antiobesity effect of ginsenoside Ro with TGR5 activation screened by a library of natural products. Our results showed that the ginsenoside Ro (90mg/kg) treatment ameliorated body weight and lipid accumulation in multiple metabolic organs of high-fat diet-induced obese (DIO) mice without affecting food intake and improved oral glucose tolerance tests, intraperitoneal insulin tolerance tests, and fasting serum glucose. We also found that triglyceride and total cholesterol in serum and liver were significantly decreased after ginsenoside Ro treatment. Then we used Tgr5 knockout mice to explore the role of Tgr5 in the antiobesity effect of ginsenoside Ro. Our results further demonstrated that ginsenoside Ro promoted glucagon-like peptide 1 (GLP-1) secretion and energy expenditure in wild-type DIO mice. However, the stimulation of ginsenoside Ro on GLP-1 secretion and energy expenditure were restrained in the Tgr5 knockout mice. In conclusion, our findings demonstrated that ginsenoside Ro ameliorates obesity and insulin resistance in DIO mice via activating TGR5, indicating a potential therapeutic role of ginsenoside Ro to treat obesity and its associated metabolic diseases. SIGNIFICANCE STATEMENT: Obesity is dramatically increasing worldwide, and it contributes to multiple metabolic diseases. G protein-coupled bile acid receptor 5 (TGR5) is a potential therapeutic target for the treatment of metabolic disorders. Ginsenoside Ro, as an oleanane-type ginsenoside, ameliorates obesity and insulin resistance, promotes glucagon-like peptide 1 secretion, and increases energy expenditure via activating TGR5. Ginsenoside Ro could be a potential leading compound for treating obesity and its associated metabolic diseases.

Cell-based high-throughput screening of polysaccharide biosynthesis hosts.

Valuable polysaccharides are usually produced using wild-type or metabolically-engineered host microbial strains through fermentation. These hosts act as cell factories that convert carbohydrates, such as monosaccharides or starch, into bioactive polysaccharides. It is desirable to develop effective in vivo high-throughput approaches to screen cells that display high-level synthesis of the desired polysaccharides. Uses of single or dual fluorophore labeling, fluorescence quenching, or biosensors are effective strategies for cell sorting of a library that can be applied during the domestication of industrial engineered strains and metabolic pathway optimization of polysaccharide synthesis in engineered cells. Meanwhile, high-throughput screening strategies using each individual whole cell as a sorting section are playing growing roles in the discovery and directed evolution of enzymes involved in polysaccharide biosynthesis, such as glycosyltransferases. These enzymes and their mutants are in high demand as tool catalysts for synthesis of saccharides in vitro and in vivo. This review provides an introduction to the methodologies of using cell-based high-throughput screening for desired polysaccharide-biosynthesizing cells, followed by a brief discussion of potential applications of these approaches in glycoengineering.

[Quantification of hepatotoxic pyrrolizidine alkaloid adonifoline in traditional Chinese medicine preparations containing Senecionis Scandentis Herba].

Pyrrolizidine alkaloids(PAs) are a kind of natural toxins, which can cause severe hepatotoxicity, pulmonary toxicity, genotoxicity, neurotoxicity, embryotoxicity and even death. Therefore, international organizations and countries such as World Health Organization have paid great attention to herbal medicines and preparations containing PAs. PAs are widely distributed in Chinese herb medicines and contained in hundreds of traditional Chinese medicine preparations. The content of adonifoline, the main PAs in Senecionis Scandentis Herba, shall be less than 0.004% in herbal medicines as described in Chinese pharmacopeia. However, there is no guidance in preparations which contain Senecionis Scandentis Herba. In this study, 14 preparations were analyzed by an UPLC-MS method. Among them, 8 preparations were found to contain adonifoline much higher than the content limits of such countries as Germany, Netherlands and New Zealand. And the highest contents of adonifoline were found in Qianbai Biyan Tablets and Qianbai Biyan Capsules, which are officially recorded in Chinese Pharmacopeia. The contents of adonifoline varied in different batches of the same preparations. The highest content was 156.10 µg·g~(-1) Qianbai Biyan Tablets, whose daily intake of adonifoline was up to 1 030.26 µg according to the recommended dosage of the preparation. Our results showed the potential risk of these preparations, and the content limit of adonifoline shall be inspected Chinese medicine preparations containing Senecionis Scandentis Herba.

The RIN-MC Fusion of MADS-Box Transcription Factors Has Transcriptional Activity and Modulates Expression of Many Ripening Genes.

Fruit development and ripening is regulated by genetic and environmental factors and is of critical importance for seed dispersal, reproduction, and fruit quality. Tomato (Solanum lycopersicum) ripening inhibitor (rin) mutant fruit have a classic ripening-inhibited phenotype, which is attributed to a genomic DNA deletion resulting in the fusion of two truncated transcription factors, RIN and MC In wild-type fruit, RIN, a MADS-box transcription factor, is a key regulator of the ripening gene expression network, with hundreds of gene targets controlling changes in color, flavor, texture, and taste during tomato fruit ripening; MC, on the other hand, has low expression in fruit, and the potential functions of the RIN-MC fusion gene in ripening remain unclear. Here, overexpression of RIN-MC in transgenic wild-type cv Ailsa Craig tomato fruits impaired several ripening processes, and down-regulating RIN-MC expression in the rin mutant was found to stimulate the normal yellow mutant fruit to produce a weak red color, suggesting a distinct negative role for RIN-MC in tomato fruit ripening. By comparative transcriptome analysis of rin and rin 35S::RIN-MC RNA interference fruits, a total of 1,168 and 1,234 genes were identified as potential targets of RIN-MC activation and inhibition. Furthermore, the RIN-MC fusion gene was shown to be translated into a chimeric transcription factor that was localized to the nucleus and was capable of protein interactions with other MADS-box factors. These results indicated that tomato RIN-MC fusion plays a negative role in ripening and encodes a chimeric transcription factor that modulates the expression of many ripening genes, thereby contributing to the rin mutant phenotype.

Cascade synthesis of uridine-5'-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes.

BACKGROUND: Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5'-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. RESULTS: To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L-1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. CONCLUSIONS: This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.

Comparison of conservative treatment outcomes for proximal humeral epiphyseal fractures in patients of different ages.

BACKGROUND: Several studies have suggested that excellent therapeutic outcomes can be achieved with conservative treatment of proximal humeral epiphyseal fractures in patients younger than 11 years old; however, the outcomes of conservative treatment for children older than 11 years are controversial. To address this problem, this study compared outcomes of conservative treatment for proximal humeral epiphyseal fractures in pediatric patients of different ages. METHODS: The patients were divided into two groups for comparative purposes based on age. Group I consisted of 34 patients who were less than 11 years old (average age: 5 years) and group II included 21 patients who were 11 years of age or older (average age: 14 years). Patients in both groups underwent conservative treatment and follow-up examination, where they first were examined with X­radiography for assessment of deformity, fracture union and loss of reduction. At the final follow-up after 2 years, patients were assessed by an interview and a detailed physical examination including the assessment of shoulder function using the Constant-Murley score. RESULTS: There were no significant differences in the grading scale of varus deformity between the two groups (P > 0.05) after immediate postreduction X­radiography; however, there were significant differences in the grading scale of varus deformity between group I and group II at the 2­year follow-up (P < 0.05). There were no significant differences between the two groups with respect to the Constant-Murley score and arm length discrepancy (P > 0.05) at final follow-up examinations. CONCLUSION: In general, the results suggested that the outcomes, as measured with radiographs, for both older and young children were comparable after immediate postreduction roentgenograms. For long-term follow-up there was a difference between the two groups and the degree of angulation and displacement might be associated with treatment outcomes for older children. Thus, these factors should be considered when treating and evaluating the outcomes for older children.

One-pot two-strain system based on glucaric acid biosensor for rapid screening of myo-inositol oxygenase mutations and glucaric acid production in recombinant cells.

The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied. In this one-pot two-strain system, GA producers-bacterial or yeast cells containing the GA biosynthetic pathway-were sorted with the help of another E. coli strain acting as a GA biosensor. The identification of highly active mutants of myo-inositol oxygenase through this system validates its effectiveness in sorting E. coli cells. Subsequently, accurate ranking of the GA synthesis capacity of a small library of Saccharomyces cerevisiae strains containing distinct GA synthesis pathways demonstrated that this optimized one-pot two-strain system may also be used for eukaryotic producer strains. These results will assist in research into metabolic engineering for GA production and development of biosensor applications.

A Viral Satellite DNA Vector (TYLCCNV) for Functional Analysis of miRNAs and siRNAs in Plants.

With experimental and bioinformatical methods, numerous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), have been found in plants, and they play vital roles in various biological regulation processes. However, most of these small RNAs remain to be functionally characterized. Until now, only several viral vectors were developed to overexpress miRNAs with limited application in plants. In this study, we report a new small RNA overexpression system via viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV) vector, which could highly overexpress not only artificial and endogenous miRNAs but also endogenous siRNAs in Nicotiana benthamiana First, we constructed basic TYLCCNV-amiRPDS(319L) vector with widely used AtMIR319a backbone, but the expected photobleaching phenotype was very weak. Second, through comparing the effect of backbones (AtMIR319a, AtMIR390a, and SlMIR159) on specificity and significance of generating small RNAs, the AtMIR390a backbone was optimally selected to construct the small RNA overexpression system. Third, through sRNA-Seq and Degradome-Seq, the small RNAs from AtMIR390a backbone in TYLCCNV-amiRPDS(390) vector were confirmed to highly overexpress amiRPDS and specifically silence targeted PDS gene. Using this system, rapid functional analysis of endogenous miRNAs and siRNAs was carried out, including miR156 and athTAS3a 5'D8(+). Meanwhile, through designing corresponding artificial miRNAs, this system could also significantly silence targeted endogenous genes and show specific phenotypes, including PDS, Su, and PCNA These results demonstrated that this small RNA overexpression system could contribute to investigating not only the function of endogenous small RNAs, but also the functional genes in plants.

Characterization of heparan sulfate N-deacetylase/N-sulfotransferase isoform 4 using synthetic oligosaccharide substrates.

BACKGROUND: The final structure of heparan sulfate chains is strictly regulated in vivo, though the biosynthesis is not guided by a template process. N-deacetylase/N-sulfotransferase (NDST) is the first modification enzyme in the HS biosynthetic pathway. The N-sulfo groups introduced by NDST are reportedly involved in determination of the susceptibility to subsequent processes catalyzed by C5-epimerse and 3-O-sulfotransferases. Understanding the substrate specificities of the four human NDST isoforms has become central to uncovering the regulatory mechanism of HS biosynthesis. METHODS: Highly-purified recombinant NDST-4 (rNDST-4) and a selective library of structurally-defined oligosaccharides were employed to determine the substrate specificity of rNDST-4. RESULTS: Full-length rNDST-4 lacks obvious N-deacetylase activity, and displays only N-sulfotransferase activity. Unlike NDST-1, NDST-4 did not show directional N-sulfotransferase activity while the N-deacetylase domain was inactive. CONCLUSION AND GENERAL SIGNIFICANCE: Individual NDST-4 could not effectively assume the key role in the distribution of N-S domains and N-Ac domains in HS biosynthesis in vivo.

KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates.

Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized. Here, we probed the substrate specificity of KfoA by a capillary electrophoresis (CE)-based method. KfoA could epimerize both acetylated and non-acetylated substrates, but its k cat/K m value for UDP-GlcNAc was approximately 1300-fold that for UDP-Glc. Recombinant KfoA showed a strong preference for acetylated substrates in vitro. The conclusion that KfoA is a higher efficiency UDP-GalNAc provider than GalE was supported by a coupled assay developed based on the donor-acceptor combination specificity of E. coli K4 chondroitin polymerase (KfoC). Furthermore, residue Ser-301, located near the UDP-GlcNAc binding pocket, plays an important role in the determination of the conversion ratio of UDP-GlcNAc to UDP-GalNAc by KfoA. Our results deepen the understanding of the mechanism of KfoA and will assist in the research into the metabolic engineering for chondroitin sulfate production.

Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum.

Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.

Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END.

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ(4)-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the ß1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-ß1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.

Small RNA profiling and degradome analysis reveal regulation of microRNA in peanut embryogenesis and early pod development.

BACKGROUND: As a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored. RESULTS: In this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively. CONCLUSIONS: Large numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

SRNAome and degradome sequencing analysis reveals specific regulation of sRNA in response to chilling injury in tomato fruit.

Plant genomes encode diverse small RNA classes that function in distinct gene-silencing pathways. To elucidate the intricate regulation of microRNAs (miRNAs) and endogenous small-interfering RNAs (siRNAs) in response to chilling injury in tomato fruit, the deep sequencing and bioinformatic methods were combined to decipher the small RNAs landscape in the control and chilling-injured groups. Except for the known miRNAs and ta-siRNAs, 85 novel miRNAs and 5 ta-siRNAs members belonging to 3 TAS families (TAS5, TAS9 and TAS10) were identified, 34 putative phased small RNAs and 740 cis/trans-natural antisense small-interfering RNAs (nat-siRNAs) were also found in our results which enriched the tomato small RNAs repository. A large number of genes targeted by those miRNAs and siRNAs were predicted to be involved in the chilling injury responsive process and five of them were verified via degradome sequencing. Based on the above results, a regulatory model that comprehensively reveals the relationships between the small RNAs and their targets was set up. This work provides a foundation for further study of the regulation of miRNAs and siRNAs in the plant in response to chilling injury.

[Design, synthesis and evaluation of a novel MEK protein inhibitors].

This study was conducted to design and synthetize highly efficient, specific, non-resistant small MEK inhibitors. Based on active small molecules which have been reported, we studied the action mode with MEK protein using Autodock 4.2, generated innovative and feasible design method, designed novel small MEK protein inhibitors with a reference to molecular modeling and docking. The anti-tumor activities of four kinds of cells including MCF-7, PANC-1, SY5Y, A549 were tested with MTT method in vitro. The structure of 10 new small molecules has been determined with 1H NMR and 13C NMR. The compounds 4, 6, 7, 8, 10 had high antitumor activities, the compounds 1, 3, 5 also showed good activity, and the compounds 2, 9 showed cell selectivity in killing tumor.