Analysis of O-acetylated sialic acids by 3-nitrophenylhydrazine derivatization combined with LC-MS/MS.

Sialic acids are a family of monosaccharides that share a nine-carbon backbone and a carboxyl group. A recent derivatization method based on 3-nitrophenylhydrazine (3-NPH) provides a mild chemical labeling technique for biomolecules containing carbonyl or carboxyl groups. In this study, we utilized 3-NPH to label sialic acids via a two-step derivatization process. The derivatized species can produce a common reporter ion corresponding to C1-C3 with two labels, and a fragment differentiating between Neu5Ac, Neu5Gc, and KDN. This method is compatible with O-acetylated sialic acids and provides high sensitivity to Neu5Gc and KDN, and since the utilization of dual labeling significantly enhances the hydrophobicity of derivatives, it can effectively mitigate matrix effects when combined with parallel reaction monitoring technology. Negative-ion tandem mass spectrometry (MS/MS) analysis reveals a distinctive fragmentation profile for the 4-O-acetylated species, while the other sialic acids yield similar MS/MS spectra with a high abundance of reporter ions. Using the reporter ion as a transition, this analytical strategy is effective for analyzing complex biological samples. For example, it was successfully employed to quantify sialic acids in the intestinal tissues of several carp species, demonstrating its potential in sialylation research.

Antagonistic mechanisms of bisphenol analogues on the estrogen receptor α in zebrafish embryos: Experimental and computational studies.

Bisphenol A (BPA) can disturb the estrogen receptor α (ERα)-mediated signaling pathway, which results in endocrine-disrupting effects and reproductive toxicity. Most BPA analogues as alternatives were evidenced to generate estrogenic activity as agonists or partial agonists of ERα. Recent studies indicated that certain BPA analogues, such as bisphenol M (BPM), bisphenol P (BPP), and bisphenol FL (BPFL), exhibited strong anti-estrogenic effects comparable with the typical antagonist 4-hydroxytamoxifen. However, conflicting findings were also observed for the compounds in different in vitro assays, and whether these BPA analogues can elicit an in vivo effect on ERα at environmentally relevant concentrations remains unknown. The underlying structural basis of estrogenic/anti-estrogenic activity should be further elucidated at the atomic level. To address these issues, we combined zebrafish-based in vivo and in silico methods to assess the effects of the compounds on ERα. The results show that the expressions of ERα-mediated downstream related genes in zebrafish embryos decreased after exposed to the compounds. Further molecular dynamics simulations were used to probe the antagonistic mechanisms of the compounds on ERα. The key H-bonding interactions were identified as important ligand recognition by ERα in the analysis of binding modes and binding free energy calculations. In summary, the current study provides preliminary in vivo evidence of fish species for the anti-estrogenic activity of certain BPA analogues.

PFOI stimulates the motility of T24 bladder cancer cells: Possible involvement and activation of lncRNA malat1.

Perfluorinated iodine alkanes (PFIs) can serve as an important raw materials for the synthesis of various perfluorinated chemical products through telomerization reaction. The estrogenic effects of PFIs have been reported previously by some in vitro and in vivo screening assays. To explore the potential epigenetic toxicity of PFIs, activation of lncRNAs was screened, and the cell motility changes induced by perfluorooctyl iodide (PFOI) were analyzed in this study. High metastatic bladder cell line (T24) was used to investigate the cellular migration function affected by PFOI. PFOI exposure significantly induced the upregulation of lncRNA anril, thorlnc, hotairm1, meg3, and malat1. The migration and invasion of T24 cells were also enhanced upon PFOI exposure. The transcription level of matrix metalloenzyme genes, epidermal growth factors, cytoskeleton genes, and the upstream factors involved in cell motility pathways were examined to illustrate possible mechanisms. Additionally, the basic role of malat1 in cellular motility was investigated by lncRNA knockdown and migration assays. The knockdown of malat1 inhibited the cellular motility induced by PFOI. The levels of MMP-2/-9 genes were also down-regulated by the treatment of si-malat1. Overall, the perturbation of cytoskeleton genes (E-cadherin/N-cadherin) may account for the impact on the motility of T24 cells. Our studies indicate that perfluorinated chemicals might regulate the lncRNAs, thus promoting the metastasis of the tumor cells.

Evaluation of hypopigmentation in embryonic zebrafish induced by emerging disinfection byproduct, 3, 5-di-I-tyrosylalanine.

Halogenated dipeptides, 3, 5-di-I-tyrosylalanine (DIYA), have been identified as novel disinfection byproducts (DBPs), following chloramination of authentic water. However, little is known about their toxicity. Zebrafish embryos were used to assess the toxicity of novel iodinated DBPs (I-DBPs). Although DIYA did not exhibit high acute toxicity to embryonic zebrafish (LC50 > 2 mM), it significantly inhibited pigmentation of melanophores and xanthophores on head, trunk and tail at 500 µM as determined by photographic analysis. Whereas N-phenylthiourea (PTU) as a pigment inhibitor did not inhibit development of yellow pigments. Colorimetric detection of melanin further confirmed these results. Quantitative real time polymerase chain reaction (qRT-PCR) measurements indicated that genes (dct, slc24a5, tyr, tyrp1a, tyrp1b, silva) associated with the melanogenesis pathway were dramatically down-regulated following exposure to 500 µM DIYA. In addition, enzymatic activity of tyrosinase (TYR) decreased, also demonstrating that the underlying mechanism of hypopigmentation was attributed to the disruption of melanogenesis pathway. Transcription levels of xanthophore genes (gch2, bnc2, csf1a, csf1b, pax7a and pax7b) were also monitored by qRT-PCR assay. DIYA exposure up-regulated expression of gch2 and bnc2, but not csf1 and pax7. Tested DIYA analogues, brominated tyrosine was unlikely to inhibit pigmentation, indicating that the iodine substitution and dipeptides structure are of important structural feature for the inhibition of pigmentation. In this study, we observed that DIYA inhibited melanogenesis related genes, which might contribute to pigmentation defects. Moreover, as an emerging I-DBPs, the developmental toxicity of aromatic dipeptides should be further studied.

Upregulation of miR150-5p in generalized myasthenia gravis patients is associated with decreased serum levels of IL-17 and increased serum levels of IL-10.

BACKGROUND: MiR150-5p has been reported to be involved in generalized myasthenia gravis, in which different cytokines play critical roles. The regulatory network of cytokines in generalized myasthenia gravis has not been fully elucidated. Our study aimed to investigate the interactions between miR150-5p and different cytokines in generalized myasthenia gravis. MATERIALS AND METHOD: Serum levels of miR150-5p and different cytokines including IL-2, IL-17, IL-10, IL-19, IL-20 and IL-35 were detected by qRT-PCR and ELISA, respectively. ROC curve analysis was performed to evaluate the diagnostic value of miR150-5p for generalized myasthenia gravis. Correlation between serum levels of miR150-5p and different cytokines were analyzed by Pearson correlation coefficient. RESULTS: Compared with healthy controls, decreased serum levels of IL-2 and IL-17 and increased serum levels of miR150-5p, IL-10, IL-19, IL-20 and IL-35 were observed in patients with generalized myasthenia gravis. Serum levels of miR150-5p were positively correlated with IL-10 and negatively correlated with IL-17. After treatments, serum levels of miR150-5p and IL-10 decreased, while serum levels of IL-2 and IL-17 increased. CONCLUSION: Upregulation of miR150-5p is involved in generalized myasthenia gravis patients and is associated with decreased serum levels of IL-17 as well as increased serum levels of IL-10.