Identification and characterization of emGalaseE, a ß-1,4 galactosidase from Elizabethkingia meningoseptica, and its application on living cell surface.

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37 °C) and pH (5-8) levels and can remove ß-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As this enzyme has a potential application for living cell glycan editing, we named it emGalaseE or glycan-editing galactosidase I (csgeGalaseI). In summary, our findings lay the groundwork for further investigation by presenting a simple and effective approach for the removal of galactose moieties from cell surface.

Computational analysis of curcumin-mediated alleviation of inflammation in periodontitis patients with experimental validation in mice.

AIM: Using network pharmacology and experimental validation to explore the therapeutic efficacy and mechanism of curcumin (Cur) in periodontitis treatment. MATERIALS AND METHODS: Network pharmacology was utilized to predict target gene interactions of Cur-Periodontitis. Molecular docking was used to investigate the binding affinity of Cur for the predicted targets. A mouse model with ligature-induced periodontitis (LIP) was used to verify the therapeutic effect of Cur. Microcomputed tomography (micro-CT) was used to evaluate alveolar bone resorption, while western blotting, haematoxylin-eosin staining and immunohistochemistry were used to analyse the change in immunopathology. SYTOX Green staining was used to assess the in vitro effect of Cur in a mouse bone marrow-isolated neutrophil model exposed to lipopolysaccharide. RESULTS: Network pharmacology identified 114 potential target genes. Enrichment analysis showed that Cur can modulate the production of neutrophil extracellular traps (NETs). Molecular docking experiments suggested that Cur effectively binds to neutrophil elastase (ELANE), peptidylarginine deiminase 4 (PAD4) and cathepsin G, three enzymes involved in NETs. In LIP mice, Cur alleviated alveolar bone resorption and reduced the expression of ELANE and PAD4 in a time-dependent but dose-independent manner. Cur can directly inhibit NET formation in the cell model. CONCLUSIONS: Our research suggested that Cur may alleviate experimental periodontitis by inhibiting NET formation.

Prognostic Value of Wagner Grade and Platelet Level in Diabetics with Infected Foot Ulcers After Antibiotic Therapy.

Purpose: The aim of the current study was to investigate factors potentially associated with the healing of infected foot ulcers in patients with diabetes after antimicrobial therapy with drugs selected based on antimicrobial susceptibility testing. Patients and Methods: A retrospective study was conducted to analyze clinical data from 99 type 2 diabetes mellitus patients with foot infection admitted to our center from January 2016 to December 2020. Pathogenic characteristics, results of wound discharge testing, and relevant wound surface factors were analyzed. Etiological characteristics and the results of susceptibility testing, wound healing rates, and factors potentially associated with wound healing rates were also analyzed. Results: Baseline data were analyzed via the t-test for independent samples, the Mann-Whitney U-test, and the chi-square test to identify variables significantly associated with prognosis. Least absolute shrinkage and selection operator regression analysis then determined that Wagner grade, essential hypertension, platelets, Gram negative bacteria, and neutrophil-to-lymphocyte ratio were of predictive value. A nomogram plot was built based on these five variables, and it yielded a standard C-index of 0.964, and an internally corrected C-index of 0.931. In multivariate logistic regression analysis Wagner grade (odds ratio [OR] 12.30, 95% confidence interval [CI] 2.471-61.194, p = 0.002) and platelet level (OR 0.978, 95% CI 0.960-0.996, p = 0.018) were significantly associated with wound healing outcomes. Restricted cubic spline analysis indicated that there was a linear relationship between wound healing and platelet levels, and that this relationship was strongest in patients classified as Wagner grade 2 with a platelet count ≤ 200 (p for nonlinearity = 0.442). Conclusion: Wagner grade, essential hypertension, platelet count, Gram negative bacteria, and neutrophil-to-lymphocyte ratio could predict the course of healing of infected foot ulcers in type 2 diabetes mellitus patients. When the Wagner grade was 2 and the platelet level was ≤ 200, platelet level was linearly associated with healing outcome, whereby a lower platelet level predicted a worse wound healing outcome.

Visceral adipose tissue increases the risk of periodontal disease: Results from the 2011-2014 National Health and Nutrition Examination Survey and Mendelian randomization analysis.

AIM: This study aimed to investigate the relationship and potential causal effect of visceral adipose tissue (VAT) on periodontal disease (PD). Despite prior research on this, there has been no definitive conclusion. Therefore, this study aimed to provide additional insight into these associations. MATERIALS AND METHODS: This study used data from the National Health and Nutrition Examination Survey 2011-2014 to examine the association between VAT and PD in a cross-sectional study design. Various analytical methods were employed, such as multivariable logistic regression, restricted cubic spline analysis, and p-value for trend. Additionally, two-sample Mendelian randomization (MR) analysis was performed to evaluate the potential causal effect of VAT on PD risk. These methods enabled us to evaluate the association between VAT and PD and to establish whether VAT could be a causal factor in PD development. RESULTS: The study examined a sample of 3535 participants, and the findings suggested that higher VAT levels were associated with an increased risk of PD. In addition, multivariable regression analysis conducted in six models revealed a statistically significant association between VAT and PD risk. Restricted cubic spline analysis showed an inverted U-shaped association between VAT and PD, with a turning point at 733 g of VAT. Finally, a two-sample MR analysis provided evidence for a potential causal relationship between VAT and PD risk, with an odds ratio of 1.16 (95% confidence interval: 1.02-1.33, p = .027) per kg increase in genetically predicted VAT. CONCLUSIONS: The results of this study suggest that there is a significant association between VAT and PD and that VAT could be a potential causal factor in PD risk. Our results also suggest that although there is a potential link between VAT levels and PD risk, the effect size is modest. Therefore, interventions designed to reduce VAT levels should not be considered a primary strategy for PD risk reduction but could be one of many strategies used in a comprehensive approach to PD risk management.

Quercetin promotes autophagy to alleviate cigarette smoke-related periodontitis.

BACKGROUND AND OBJECTIVES: Cigarette smoking has been reported as an independent risk factor for periodontitis. Tobacco toxins affect periodontal tissue not only locally but also systemically, leading to the deterioration and recurrence of periodontitis. However, the mechanism of cigarette smoke-related periodontitis (CSRP) is unclear and thus lacks targeted treatment strategies. Quercetin, a plant-derived polyphenolic flavonoid, has been reported to have therapeutic effects on periodontitis due to its documented antioxidant activity. This study aimed to evaluate the effects of quercetin on CSRP and elucidated the underlying mechanism. METHODS: The cigarette smoke-related ligature-induced periodontitis mouse model was established by intraperitoneal injection of cigarette smoke extract (CSE) and silk ligation of bilateral maxillary second molars. Quercetin was adopted by gavage as a therapeutic strategy. Micro-computed tomography was used to evaluate the alveolar bone resorption. Immunohistochemistry detected the oxidative stress and autophagy markers in vivo. Cell viability was determined by Cell Counting Kit-8, and oxidative stress levels were tested by 2,7-dichlorodihydrofluorescein diacetate probe and lipid peroxidation malondialdehyde assay kit. Alkaline phosphatase and alizarin red staining were used to determine osteogenic differentiation. Network pharmacology analysis, molecular docking, and western blot were utilized to elucidate the underlying molecular mechanism. RESULTS: Alveolar bone resorption was exacerbated and oxidative stress products were accumulated during CSE exposure in vivo. Oxidative stress damage induced by CSE caused inhibition of osteogenic differentiation in vitro. Quercetin effectively protected the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and periodontal tissue by upregulating the expression of Beclin-1 thus to promote autophagy and reduce oxidative stress damage. CONCLUSION: Our results established a role of oxidative stress damage and autophagy dysfunction in the mechanism of CSE-induced destruction of periodontal tissue and hPDLCs, and provided a potential application value of quercetin to ameliorate CSRP.

Three-Dimensional Matrix Stiffness Activates the Piezo1-AMPK-Autophagy Axis to Regulate the Cellular Osteogenic Differentiation.

Extracellular matrix (ECM) stiffness is a key stimulus affecting cellular differentiation, and osteoblasts are also in a three-dimensional (3D) stiff environment during the formation of bone tissues. However, it remains unclear how cells perceive matrix mechanical stiffness stimuli and translate them into intracellular signals to affect differentiation. Here, for the first time, we constructed a 3D culture environment by GelMA hydrogels with different amino substitution degrees and found that Piezo1 expression was significantly stimulated by the stiff matrix with high substitution; meanwhile, the expressions of osteogenic markers OSX, RUNX2, and ALP were also observably improved. Moreover, knockdown of Piezo1 in the stiff matrix revealed significant reduction of the abovementioned osteogenic markers. In addition, in this 3D biomimetic ECM, we also observed that Piezo1 can be activated by the static mechanical conditions of the stiff matrix, leading to the increase of the intracellular calcium content and accompanied with a continuous change in cellular energy levels as ATP was consumed during cellular differentiation. More surprisingly, we found that in the 3D stiff matrix, intracellular calcium as a second messenger promoted the activation of the AMP-activated protein kinase (AMPK) and unc-51-like autophagy-activated kinase 1 (ULK1) axis and modestly modulated the level of autophagy, bringing it more similar to differentiated osteoblasts, with increased ATP energy metabolism consumption. Our study innovatively clarifies the regulatory role of the mechanosensitive ion channel Piezo1 in a static mechanical environment on cellular differentiation and verifies the activation of the AMPK-ULK1 axis in the cellular ATP energy metabolism and autophagy level. Collectively, our research develops the understanding of the interaction mechanisms of biomimetic extracellular matrix biomaterials and cells from a novel perspective and provides a theoretical basis for bone regeneration biomaterials design and application.

Identification and characterization of an α-1,3 mannosidase from Elizabethkingia meningoseptica and its potential attenuation impact on allergy associated with cross-reactive carbohydrate determinant.

Core α-1,3 mannose is structurally near the core xylose and core fucose on core pentasaccharide from plant and insect glycoproteins. Mannosidase is a useful tool for characterization the role of core α-1,3 mannose in the composition of glycan related epitope, especially for those epitopes in which core xylose and core fucose are involved. Through functional genomic analysis, we identified a glycoprotein α-1,3 mannosidase and named it MA3. We used MA3 to treat allergen horseradish peroxidase (HRP) and phospholipase A2 (PLA2) separately. The results showed that after MA3 removed α-1,3 mannose on HRP, the reactivity of HRP with anti-core xylose polyclonal antibody almost disappeared. And the reactivity of MA3-treated PLA2 with anti-core fucose polyclonal antibody decreased partially. In addition, when PLA2 was conducted enzyme digestion by MA3, the reactivity between PLA2 and allergic patients' sera diminished. These results demonstrated that α-1,3 mannose was an critical component of glycan related epitope.

Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis - sodium dodecyl sulfate, capillary zone electrophoresis - mass spectrometry and capillary isoelectric focusing - mass spectrometry.

Critical quality attributes (CQAs) of recombinant monoclonal antibody therapeutics are constantly monitored throughout the life cycle of drug development and manufacturing. In the past few decades, numerous analytical techniques have been developed for the characterization of CQAs. In this regard, non-reduced and reduced capillary electrophoresis - sodium dodecyl sulfate (CE-SDS) methods have been widely adopted by the biopharmaceutical industry for the evaluation of size-related heterogeneities in biologics. In this work we demonstrate that, with recent development of capillary electrophoresis - mass spectrometry (CE-MS) technologies, a clipping variant of bevacizumab may be identified directly by both capillary zone electrophoresis - mass spectrometry (CZE-MS) and capillary isoelectric focusing - mass spectrometry (cIEF-MS) approaches, providing a powerful addition to the traditional CE-SDS analysis workflow. In this novel workflow, linear regression between the mobility and molecular weight first results in an approximate size range of this variant. The intact masses of all species in the bevacizumab are then obtained, after deconvolution of all features identified in the CZE-MS analysis. Subsequent  CZE-MS analysis of the subunits of bevacizumab leads to the confirmation of a clipped heavy chain. Furthermore, cIEF-MS of the intact bevacizumab confirms the existence of this clipping variant. The cross-validation between CE-SDS, CZE-MS, and cIEF-MS, creates a comprehensive roadmap for monoclonal antibody size variants profiling. These CE-based analytical techniques are complementary to each other, leading to orthogonal verification for size heterogeneity characterization.

Single Cell Raman Spectroscopy Deuterium Isotope Probing for Rapid Antimicrobial Susceptibility Test of Elizabethkingia spp.

Nosocomial infection by multi-drug resistance Elizabethkingia spp. is an emerging concern with severe clinical consequences, particularly in immunocompromised individuals and infants. Efficient control of this infection requires quick and reliable methods to determine the appropriate drugs for treatment. In this study, a total of 31 Elizabethkingia spp., including two standard strains (ATCC 13253 and FMS-007) and 29 clinical isolates obtained from hospitals in China were subjected to single cell Raman spectroscopy analysis coupled with deuterium probing (single cell Raman-DIP). The results demonstrated that single cell Raman-DIP could determine antimicrobial susceptibility of Elizabethkingia spp. in 4 h, only one third of the time required by standard broth microdilution method. The method could be integrated into current clinical protocol for sepsis and halve the report time. The study also confirmed that minocycline and levofloxacin are the first-line antimicrobials for Elizabethkingia spp. infection.

LMCD1 antisense RNA 1 (LMCD1-AS1) potentiates thyroid cancer cell growth and stemness via a positive feedback loop of LMCD1-AS1/miR-1287-5p/GLI2.

BACKGROUND: LMCD1 antisense RNA 1 (LMCD1-AS1) is a certified oncogene in several tumour types. However, its role in thyroid cancer (THCA) remains unknown. METHODS: The expression level of LMCD1-AS1 in THCA cells and the normal control cell was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of LMCD1-AS1 knockdown on cell proliferation, migration and apoptosis were detected by colony formation assay, EdU assay, wound healing assay and TUNEL assay. Sphere formation assay was applied to assess sphere formation ability of THCA cells. Bioinformatics analysis and mechanism experiments, including ChIP assay, RIP assay and luciferase reporter assay were conducted to evaluate the downstream and upstream molecular mechanisms of LMCD-AS1. RESULTS: A marked up-regulation of LMCD1-AS1 in THCA cells relative to normal control cells was found. LMCD1-AS1 silencing suppressed proliferation and migration but induced apoptosis in THCA cells. Moreover, LMCD1-AS1 knockdown reduced the sphere formation capacity of THCA cells. The transcriptional factor GLI family zinc finger 2 (GLI2) binds to LMCD1-AS1, which contributed to LMCD1-AS1 up-regulation in THCA cells. Cytoplasmic LMCD1-AS1 sponged a shared microRNA between LMCD1-AS1 and GLI2. GLI2 was inhibited bymiR-1287-5p and disinhibited by LMCD1-AS1. CONCLUSIONS: LMCD1-AS1exerts pro-tumorigenic function through sponging miR-1287-5p to elevate GLI2 expression in THCA development, constituting a feedback loop of LMCD1-AS1/miR-1287-5p/GLI2.

Growth inhibition by bacterial Cas2Em proteins expressed in mammalian cells.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) proteins are bacterial adaptive immune system for survival. In our previous study, we demonstrate that polyploid giant bacterial cells (PGBC) induced by Cas2 protein is a step required by new spacer acquisition reaction catalyzed by Cas1/Cas2 complex. We also demonstrated that a carboxyl terminal domain on Cas2Em (the protein Cas2 cloned from Elizabethkingia meningoseptica) is sufficient and enough for PGBC. Thus, the potential role of Cas2Em in microbial-host interaction was explored in this study. METHODS: The impacts of Cas2Em on growth of both CHO-K1 and Hela cells were investigated. The subcellular localization and potential molecular target of Ca2Em were studied. RESULTS: The growth of mammalian cells were inhibited by Cas2Em protein via G1 arresting and apoptosis. In addition, we also demonstrated that Cas2Em was tightly associated with nuclear outer membrane and could be immunoprecipitated with 14-3-3γ through a 30 amino acid domain (homology of CLK2). CONCLUSION: Cas2Em significantly suppressed the growth of mammalian cells indicating Cas2 proteins play an important role in mammalian cells.

Filamentation initiated by Cas2 and its association with the acquisition process in cells.

Cas1-and-Cas2-mediated new spacer acquisition is an essential process for bacterial adaptive immunity. The process is critical for the ecology of the oral microflora and oral health. Although molecular mechanisms for spacer acquisition are known, it has never been established if this process is associated with the morphological changes of bacteria. In this study, we demonstrated a novel Cas2-induced filamentation phenotype in E. coli that was regulated by co-expression of the Cas1 protein. A 30 amino acid motif at the carboxyl terminus of Cas2 is necessary for this function. By imaging analysis, we provided evidence to argue that Cas-induced filamentation is a step coupled with new spacer acquisition during which filaments are characterised by polyploidy with asymmetric cell division. This work may open new opportunities to investigate the adaptive immune response and microbial balance for oral health.

Identification and characterization of a novel glycoprotein core xylosidase from the bacterium Elizabethkingia meningoseptica.

Although core xylose on glycoproteins has been implicated in allergy, infection and other biological processes, research on core xylose modification is rare. The lack of a ß-d-xylosidase that can catalytically remove the core xylose directly from glycoproteins is a reason for this. Through functional genomic analysis, we identified a glycoprotein core xylosidase and named it gpcXase I. gpcXase I is located immediately downstream of glycoprotein core fucosidase cFase I in Elizabethkingia meningoseptica. These two genes form a functional operon for glycoprotein core modifications. Three acidic residues (Asp-200, Asp-304 and Glu-649) were identified as key catalytic sites for gpcXase I activity, suggesting a unique triacdic mechanize for its activity. Asp-200 was identified a novel and essential base catalysts in the catalytic process, Asp-304 and Glu-649 was function as catalytic nucleophiles and acid catalysts, respectively. In addition, IgE-specific reactions were detected in 55% of serum samples collected from 40 allergic patients, and the reactions were significantly attenuated by removal of the core xylose of the allergen by treatment with gpcXase I. gpcXase I is a novel tool for basic and clinical glycomics.

Reaction-based phosphorescent nanosensor for ratiometric and time-resolved luminescence imaging of fluoride in live cells.

A two-channel phosphorescent nanosensor for fluoride with excellent selectivity and sensitivity has been designed and synthesized. By using the specific chemical affinity between silicon and fluoride, the nanosensor has been used for ratiometric and time-resolved luminescence detection of F(-) in aqueous media and live cells.

Nodularins in poisoning.

Nodularins are an important class of hepatotoxic cyclic pentapeptides that are produced by the cyanobacteria Nodularia spumigena. These peptides have been found worldwide and have been implicated in the deaths of animals as well as a potent cyanotoxin in humans. To date, approximately 10 variants have been discovered, among which nodularin-R is the most abundant. Though the mechanisms of their potential hepatotoxicity and carcinogenicity are not well understood, the most frequently proposed mechanisms are described here. Most importantly, a comprehensive review of nodularins in poisoning is presented, including their bioaccumulation in water, cyanobacterial blooms and aquatic animals, the IC50, LC50 and LD50 values determined in laboratories, and wild, domestic and laboratory animal cases. However, the hazard of these toxins to humans has not been fully elucidated, predominantly due to the lack of exposure data. One of reasons underlying is that most current methods are ill suited for clinical monitoring. Thus, the available assays for the detection and quantification of nodularins are described with an emphasis on the problems encountered with each assay. Our ultimate aim is to demonstrate the urgency of better understanding the toxicity of nodularins, especially in humans, and thus effectively protecting ourselves from their poisoning.

2-[3-(2-Acetoxyphenyl)quinoxa-lin-2-yl]phenyl acetate.

The title compound, C24H18N2O4, crystallizes as a syn-conformer, with dihedral angles between the quinoxaline moiety and the acet-oxy-substituted benzene rings of 53.46 (3)° and 54.78 (3)°. In the crystal, the mol-ecules form chains along [100] via C-H⋯O inter-actions.

Highly efficient vinylaromatics generation via iron-catalyzed sp3 C-H bond functionalization CDC reaction: a novel approach to preparing substituted benzo[α]phenazines.

An iron-catalyzed benzylic vinylation was developed to transfer the carbon atom in the N,N-dimethyl moiety of N,N-dimethylacetamide (or N,N-dimethylformamide) to 2-methyl azaarenes to generate 2-vinyl azaarenes.