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The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O -glycans, but not the N -glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N -glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N -glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N -glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N -glycans and O -glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N -glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N - and O -glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Chondroitin sulfate (CS) has an important role in cell division, in the central nervous system, and in joint-related pathologies such as osteoarthritis. Due to the complex chemical structure and biological importance of CS, simple, sensitive, high resolution, and robust analytical methods are needed for the analysis of CS disaccharides and oligosaccharides. An ion-pairing, reversed-phase, ultraperformance liquid chromatography (IPRP–UPLC) separation, coupled to electrospray ionization mass spectrometry with an ion trap mass analyzer, was applied for the analyses of CS-derived disaccharides. UPLC separation technology uses small particle diameter, short column length, and elevated column temperature to obtain high resolution and sensitivity. Hexylamine (15mM) was selected as the optimal ion-pairing reagent. [Copyright &y& Elsevier]

Abstract: Liquid chromatography–mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A. [Copyright &y& Elsevier]

Abstract: Traditional chromatographic quantification methods for heparosan produced from the Escherichia coli K5 strain rely on extensive purification requiring laborious sample preparation. These methods are time-consuming, often resulting in sample loss during purification, and thus might not accurately reflect the amount of heparosan in the original mixture. A simple, sensitive 1H nuclear magnetic resonance (NMR) quantification method that directly quantifies heparosan K5 polysaccharide present in E. coli fermentation supernatant is described. [Copyright &y& Elsevier]

Anthocyanins in different types of black rice are different and their in vivo antioxidant activity is poorly studied. In this study, nine anthocyanins were detected by high performance liquid chromatography (HPLC) and eight of them were identified by ultra performance liquid chromatography/time-of-flight mass spectrometry (UPLC/Q-TOF-MS) in black rice. The in vivo antioxidant activity of the black rice extract containing 41.69% of anthocyanins was determined by the model of KBrO 3 -induced renal injury in mice. KBrO 3 caused serious renal injury, with a significant increase in serum creatinine (14.37 ± 0.27 μg/mL versus 8.80 ± 0.38 μg/mL) and blood urea nitrogen (BUN, 625.56 ± 42.62 mg/L versus 348.49 ± 14.90 mg/L) levels. KBrO 3 could also cause oxidative stress in the kidney, with a significant increase in renal xanthine oxidase (XOD), malondialdehyde (MDA) and nitric oxide (NO) levels. Pretreatment with the anthocyanin-rich black rice extract remarkably reduced the levels of serum creatinine and BUN and the levels of renal XOD, MDA and NO. The protective effects were due to the free radical scavenging capacity of the anthocyanins. [ABSTRACT FROM AUTHOR]

• Large proportions of inpatients with COVID-19 had mental health problems. • Sex and levels of inflammatory markers were related to mental health symptoms. • Self-perceived illness severity mediated effect of disease duration on mental health. To evaluate the mental health status of hospitalized patients with coronavirus disease 2019 (COVID-19) and to explore the related factors. This was a cross-sectional survey among COVID-19 inpatients in two isolation wards of a designated hospital in Wuhan, China, from March 7, 2020, to March 24, 2020. Participants' demographic data, clinical data and levels of circulating inflammatory markers were collated. Mental health symptoms were evaluated with questionnaires, which included the Insomnia Severity Index (ISI) scale, the 9-item Patient Health Questionnaire (PHQ-9), the 7-item Generalized Anxiety Disorder (GAD-7) scale, and questions about patients' self-perceived illness severity. Multivariate linear regression analysis was performed to explore factors that associated with mental symptoms, and a structural equation model (SEM) was used to assess the possible relationships between those factors and the patients' mental health. Among the 85 participants, 45.9% had symptoms of depression (PHQ-9 ≥ 5), 38.8% had anxiety (GAD-7 ≥ 5), and 54.1% had insomnia (ISI ≥ 8). According to multivariate regression analysis, female sex, a higher level of interleukin (IL)-1β and greater self-perceived illness severity were all significantly associated with a higher PHQ-9 score, higher GAD-7 score and higher ISI score. In addition, the disease duration and the neutrophil to lymphocyte ratio (NLR) were positively related to patients' self-perceived illness severity. The results of the SEM analyses suggested that sex (β = 0.313, P < 0.001), self-perceived illness severity (β = 0.411, P < 0.001) and levels of inflammatory markers (β = 0.358, P = 0.002) had direct effects on patients' mental health. The disease duration (β = 0.163, P = 0.003) and levels of inflammatory markers (β = 0.101, P = 0.016) also indirectly affected patients' mental health, with self-perceived illness severity acting as a mediator. A majority of COVID-19 infected inpatients reported experiencing mental health disturbances. Female sex, disease duration, levels of inflammatory markers and self-perceived illness severity are factors that could be used to predict the severity of patients' mental symptoms. [ABSTRACT FROM AUTHOR]

Aconitine (AC) is a highly toxic alkaloid from bioactive plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. In this study, we systematically evaluated the potential role of P-glycoprotein (P-gp) in the mechanisms underlying the low and variable bioavailability of oral AC. First, the bidirectional transport of AC across Caco-2 and MDCKII-MDR1 cells was investigated. The efflux of AC across monolayers of these two cell lines was greater than its influx. Additionally, the P-gp inhibitors, verapamil and cyclosporin A, significantly decreased the efflux of AC. An in situ intestinal perfusion study in rats showed that verapamil co-perfusion caused a significant increase in the intestinal permeability of AC, from 0.22×10−5 to 2.85×10−5 cm/s. Then, the pharmacokinetic profile of orally administered AC with or without pre-treatment with verapamil was determined in rats. With pre-treatment of verapamil, the maximum plasma concentration (Cmax) of AC increased sharply, from 39.43 to 1490.7ng/ml. Accordingly, a 6.7-fold increase in the area under the plasma concentration–time curve (AUC0–12h) of AC was observed when co-administered with verapamil. In silico docking analyses suggested that AC and verapamil possess similar P-gp recognition mechanisms. This work demonstrated that P-gp is involved in limiting the intestinal absorption of AC and attenuating its toxicity to humans. Our data indicate that potential P-gp-mediated drug–drug interactions should be considered carefully in the clinical application of aconite and formulations containing AC. [ABSTRACT FROM AUTHOR]