Production, purification, characterization, and safety evaluation of constructed recombinant D-psicose 3-epimerase.

BACKGROUND: D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose, is a low-calorie sweetener that has gained considerable attention as a healthy alternative sweetener due to its notable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, investigated the enzymatic characterization, and then optimized the D-psicose production. Finally, the produced D-psicose syrup underwent acute toxicity evaluation to provide scientific evidence supporting its safety. RESULTS: The optimization of DPEase expression involved the utilization of Mn2+ as a cofactor, fine-tuning isopropyl ß-D-1-thiogalactopyranoside induction, and controlling the induction temperature. The purification process was strategically designed by a nickel column and an elution buffer containing 200 mM imidazole, resulting in purified DPEase with a notable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions were at pH 7.5 and 55 °C with a final concentration of 10 mM Mn2+ addition using purified DPEase to achieve the highest D-psicose concentration of 5.60% (w/v) using 25% (w/v) of fructose concentration with a conversion rate of 22.42%. Kinetic parameters of the purified DPEase were Vmax and Km values of 28.01 mM/min and 110 mM, respectively, which demonstrated the high substrate affinity and efficiency of DPEase conversion by the binding site of the fructose-DPEase-Mn2+ structure. Strategies for maintaining stability of DPEase activity were glycerol addition and storage at -20 °C. Based on the results from the acute toxicity study, there was no toxicity to rats, supporting the safety of the mixed D-fructose-D-psicose syrup produced using recombinant DPEase. CONCLUSIONS: These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. This research should advance the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.

The transcriptional regulator Fur modulates the expression of uge, a gene essential for the core lipopolysaccharide biosynthesis in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium. RESULTS: Using bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels. CONCLUSIONS: Here, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.

The Formation of D-Allulose 3-Epimerase Hybrid Nanoflowers and Co-Immobilization on Resins for Improved Enzyme Activity, Stability, and Processability.

As a low-calorie sugar, D-allulose is produced from D-fructose catalyzed by D-allulose 3-epimerase (DAE). Here, to improve the catalytic activity, stability, and processability of DAE, we reported a novel method by forming organic-inorganic hybrid nanoflowers (NF-DAEs) and co-immobilizing them on resins to form composites (Re-NF-DAEs). NF-DAEs were prepared by combining DAE with metal ions (Co2+, Cu2+, Zn2+, Ca2+, Ni2+, Fe2+, and Fe3+) in PBS buffer, and were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. All of the NF-DAEs showed higher catalytic activities than free DAE, and the NF-DAE with Ni2+ (NF-DAE-Ni) reached the highest relative activity of 218%. The NF-DAEs improved the thermal stability of DAE, and the longest half-life reached 228 min for NF-DAE-Co compared with 105 min for the free DAE at 55 °C. To further improve the recycling performance of the NF-DAEs in practical applications, we combined resins and NF-DAEs to form Re-NF-DAEs. Resins and NF-DAEs co-effected the performance of the composites, and ReA (LXTE-606 neutral hydrophobic epoxy-based polypropylene macroreticular resins)-based composites (ReA-NF-DAEs) exhibited outstanding relative activities, thermal stabilities, storage stabilities, and processabilities. The ReA-NF-DAEs were able to be reused to catalyze the conversion from D-fructose to D-allulose, and kept more than 60% of their activities after eight cycles.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production.

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.

Enhancing the stability of a novel D-allulose 3-epimerase from Ruminococcus sp. CAG55 by interface interaction engineering and terminally attached a self-assembling peptide.

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡ = 54 kJ mol-1), significant loss in entropy ( - T Δ S ‡ = 20 kJ mol-1; 298 K) and negative activation heat capacity ( Δ C p ‡ = -0.64 kJ mol-1 K-1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10 µM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

Rational design improves both thermostability and activity of a new D-tagatose 3-epimerase from Kroppenstedtia eburnean to produce D-allulose.

D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency. Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds, and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; Tm value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

A mutation in CsGME encoding GDP-mannose 3,5-epimerase results in little and wrinkled leaf in cucumber.

KEY MESSAGE: Map-based cloning revealed that a mutation in a highly conserved amino acid of the CsGME gene encoding GDP-mannose 3,5-epimerase, causes the phenotype of little and wrinkled leaves in cucumbers. Leaf size is a critical determinant of plant architecture in cucumbers, yet only a few genes associated with this trait have been mapped or cloned. Here, we identified and characterized a mutant with little and wrinkled leaves, named lwl-1. Genetic analysis revealed that the phenotype of the lwl-1 was controlled by a single recessive gene. Through map-based cloning, the lwl-1 locus was narrowed down to a 12.22-kb region exclusively containing one fully annotated gene CsGME (CsaV3_2G004170). CsGME encodes GDP-mannose 3,5-epimerase, which is involved in the synthesis of ascorbic acid (ASA) and one of the components of pectin, RG-II. Whole-length sequencing of the 12.22 kb DNA fragment revealed the presence of only a non-synonymous mutation located in the sixth exon of CsGME in lwl-1, resulting in an amino acid alteration from Pro363 to Leu363. This mutation was unique among 118 inbred lines from cucumber natural populations. CsGME expression significantly reduced in various organs of lwl-1, accompanied by a significant decrease in ASA and pectin content in leaves. Both CsGME and Csgme proteins were localized to the cytoplasm. The mutant phenotype exhibited partial recovery after the application of exogenous boric acid. Silencing CsGME in cucumber through VIGS confirmed its role as the causal gene for lwl-1. Transcriptome profiling revealed that CsGME greatly affected the expression of genes related to the cell division process and cell plate formation. This study represents the first report to characterize and clone the CsGME in cucumber, indicating its crucial role in regulating leaf size and development.

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Characterization and expression of highly active recombinant human glucuronyl C5-epimerase in mammalian cells.

Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N-sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N-linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi.

FUCA2 and TSTA3 expression in gastric cancer: candidate biomarkers of malignant transformation.

INTRODUCTION: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC. MATERIALS AND METHODS: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters. RESULTS: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125). CONCLUSIONS: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.

Comprehensive bioinformatics analysis confirms RBMS3 as the central candidate biological target for ovarian cancer.

Ovarian cancer (OC) is one of the most lethal malignancies in the female reproductive system. To find genes related to cancer progression targeting specific biological factors for targeted therapy, bioinformatics technology has been widely used. To screen the prognostic gene markers of OC by bioinformatics and explore their potential molecular biological mechanisms. Two data sets related to OC, GSE54388, and GSE119056, were rooted in the open comprehensive gene expression database (GEO). To correct the background of the data, standardize and screen differentially expressed genes (DEGs) using the R software limma package. The selected DEGs were enriched by Gene Ontology (GO) and through DAVID online database. Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis and protein-protein interaction network (PPI-network) map were constructed by STRING online database and Cytoscape software. Combined with the TCGA database, univariate and multivariate COX regression were used to screen prognostic genes. QRT-PCR was used to verify DEGs in clinical tissue samples. Eventually, the function of RBMS3 on the viability, migration, invasion, and apoptosis of OC cells was tested through functional experiments in vitro. 352 common DEGs were screened from GSE54388 and GSE119056 data sets. Survival analysis showed that MEIS2, TSTA3, CNTN1, RBMS3, and TRA2A were considered to be connected with the prognosis of OC. We discover that the expression level of RBMS3 was positively connected with the overall survival (OS) rate of sufferers with OC. The level of RBMS3 in OC tissues was markedly lower than that in neighboring structures and the outcomes of the GEPIA database were consistent with those of the qRT-PCR experiment. Through gene transfection technology it was found that overexpression of RBMS3 in OC cells substantially suppressed the vitality, migration, and invasion of OC cells and raised the rates of apoptosis in the OC cells. In this experiment, we distinguish 5 genes that may participate in the prognosis of OC and showed the key genes and pathways related to OC. It is speculated that RBMS3, a tumor suppressor gene, can be applied as a potential biological marker for the treatment of OC, gene expression summary, and prognosis.

Designing a Highly Efficient Biosynthetic Route for Lacto-N-Neotetraose Production in Escherichia coli.

Recently, the biosynthesis of human milk oligosaccharides (HMOs) has been attracting increasing attention. Lacto-N-neotetraose (LNnT) is one of the most important neutral-core HMOs with promising health effects for infants. It has received Generally Recognized as Safe (GRAS) status and is the second HMO commercially added in infant formula after 2'-fucosyllactose. In previous studies, a series of engineered Escherichia coli strains have been constructed and optimized to produce high titers of precursor lacto-N-triose II. On the basis of these strains, LNnT-producing strains were constructed by overexpressing the ß1,4-galactosyltransferase-encoding gene from Aggregatibacter actinomycetemcomitans NUM4039 (Aa-ß1,4-GalT). Interestingly, an appreciable LNnT titer was obtained by weakening the metabolic flux of the UDP-GlcNAc pathway and simply overexpressing the essential genes lgtA, galE, and Aa-ß1,4-GalT in lacZ-, wecB-, and nagB-deleted E. coli. Subsequently, LNnT synthesis was optimized through balancing the expression of these three biosynthetic enzymes. The optimized strain produced LNnT with an extracellular titer of 12.1 g/L in fed-batch cultivation, with the productivity and specific yield of 0.25 g/L·h and 0.27 g/g dry cell weight, respectively.

Eriodictyol suppresses the malignant progression of colorectal cancer by downregulating tissue specific transplantation antigen P35B (TSTA3) expression to restrain fucosylation.

Eriodictyol is a natural flavonoid with many pharmacological effects, such as anti-oxidation, anti-inflammation, anti-tumor, and neuroprotection. Besides, it has been reported that flavonoids play an important role in protein glycosylation. The fucosylation structure is closely associated with processes of various tumor metastases. TSTA3 is involved in the de novo synthesis and can convert cellular GDP-D-mannose into GDP-L-fucose. It was predicted on the STITCH database that eriodictyol interacted with TSTA3. In addition, literature has confirmed that TSTA3 is upregulated in CRC and can regulate the proliferation and migration of breast cancer cells. Herein, the precise effects of eriodictyol on the clone-forming, proliferative, migratory and invasive abilities of CRC cells as well as EMT process were assessed. Moreover, the correlation among eriodictyol, TSTA3, and fucosylation in these malignant behaviors of CRC cells was evaluated, in order to elucidate the underlying mechanism. The current work discovered that eriodictyol inhibited the viability, clone-formation, proliferation, migration, invasion, and EMT of CRC cells, and that these inhibitory effects of eriodictyol on the malignant behavior of CRC cells were reversed by TSTA3 overexpression. Additionally, eriodictyol suppresses fucosylation by downregulating the TSTA3 expression. Results confirmed that fucosylation inhibitor (2-F-Fuc) inhibited clone formation, proliferation, migration, invasion, as well as EMT of CRC cells and eriodictyol treatment further reinforced the suppressing effects of 2-F-Fuc on the malignant behavior of CRC cells. We conclude that eriodictyol suppresses the clone-forming, proliferative, migrative and invasive abilities of CRC cells as well as represses the EMT process by downregulating TSTA3 expression to restrain fucosylation.

Intrinsic Expression of Coagulation Factors and Protease Activated Receptor 1 (PAR1) in Photoreceptors and Inner Retinal Layers.

The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1-/- mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function.

The ß-lactam ticarcillin is a Staphylococcus aureus UDP-N-acetylglucosamine 2-epimerase binder.

Infectious diseases account for 25% of the causes of death worldwide and this rate is expected to increase due to antibiotic resistance. Among the bacteria associated with healthcare infections, Staphylococcus aureus is a prevalent pathogen and about 50% of the isolates are found to be methicillin-resistant. Here we describe the identification of ticarcillin as a weak binder of the S. aureus UDP-N-acetylglucosamine 2-epimerase. After a docking screening, ticarcillin was identified as a ligand in using the recently proposed isothermal analysis of differential scanning fluorimetry data. Finally, an equilibrium MD simulation confirmed the docking binding mode as a stable pose, with large contributions to the binding energy coming from interactions between Arg206 and Arg207 and the carboxylate groups in ticarcillin.