Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.

Amino acids and wound healing in people with limb-threatening diabetic foot ulcers.

BACKGROUND: Amino acids are associated with wound healing in traumatic wounds and burns, although their effects on healing in patients with diabetic foot ulcers (DFUs) are limited. This study aimed to evaluate and identify specific amino acids associated with healing outcomes of patients with DFUs. METHODS: Sixty-two out of 85 patients who completed the in-hospital treatment for limb-threatening DFUs were enrolled. All ulcers had epithelialization without clinical evidence of infection at discharge. The patients and their families were instructed on foot-care techniques and committed to regular follow-up for 1 year. Baseline characteristics, PEDIS wound classification, laboratory data and serum amino acid levels were used to analyze their predictive power. RESULTS: Fifty-seven patients completed the study in which 38 had healed and 19 had unhealed ulcers. The unhealed group had higher incidence of coronary artery disease and larger wound size. Most patients received endovascular therapy (81.6% healed group; 78.9% unhealed group) before enrollment. Following adjustments for clinical factors, the serum levels of arginine (326.4 µmol/L vs. 245.0 µmol/L, P = 0.045), isoleucine (166.7 µmol/L vs. 130.1 µmol/L, P = 0.019), leucine (325.8 µmol/L vs. 248.9 µmol/L, P = 0.039), and threonine (186.7 µmol/L vs. 152.0 µmol/L, P = 0.019) were significantly higher in the healed group. CONCLUSIONS: The amino acids associated with wound healing in DFUs differ from those reported for traditional traumatic wounds. These findings affirm the necessity for future large-scaled studies for the application of these amino acids in DFU healing, either as prognostic predictors or supplemented regimens.

Aeginetia indica Decoction Inhibits Hepatitis C Virus Life Cycle.

Chronic hepatitis C virus (HCV) infection is still a global epidemic despite the introduction of several highly effective direct-acting antivirals that are tagged with sky-high prices. The present study aimed to identify an herbal decoction that ameliorates HCV infection. Among six herbal decoctions tested, the Aeginetia indica decoction had the most profound effect on the HCV reporter activity in infected Huh7.5.1 liver cells in a dose- and time-dependent manner. The Aeginetia indica decoction exerted multiple inhibitory effects on the HCV life cycle. Pretreatment of the cells with the Aeginetia indica decoction prior to HCV infection reduced the HCV RNA and non-structural protein 3 (NS3) protein levels in the infected cells. The Aeginetia indica decoction reduced HCV internal ribosome entry site-mediated protein translation activity. It also reduced the HCV RNA level in the infected cells in association with reduced NS5A phosphorylation at serine 235, a predominant phosphorylation event indispensable to HCV replication. Thus, the Aeginetia indica decoction inhibits HCV infection, translation, and replication. Mechanistically, the Aeginetia indica decoction probably reduced HCV replication via reducing NS5A phosphorylation at serine 235.

Dietary flavonoids, luteolin and quercetin, inhibit invasion of cervical cancer by reduction of UBE2S through epithelial-mesenchymal transition signaling.

We previously reported that the dietary flavonoids, luteolin and quercetin, might inhibit the invasiveness of cervical cancer by reversing epithelial-mesenchymal transition (EMT) signaling. However, the regulatory mechanism exerted by luteolin and quercetin is still unclear. This study analyzed the invasiveness activation by ubiquitin E2S ligase (UBE2S) through EMT signaling and inhibition by luteolin and quercetin. We found that UBE2S expression was significantly higher in highly invasive A431 subgroup III (A431-III) than A431-parental (A431-P) cells. UBE2S small interfering (si)RNA knockdown and overexpression experiments showed that UBE2S increased the migratory and invasive abilities of cancer cells through EMT signaling. Luteolin and quercetin significantly inhibited UBE2S expression. UBE2S showed a negative correlation with von Hippel-Lindau (VHL) and a positive correlation with hypoxia-induced factor (Hif)-1α. Our findings suggest that high UBE2S in malignant cancers contributes to cell motility through EMT signaling and is reversed by luteolin and quercetin. UBE2S might contribute to Hif-1α signaling in cervical cancer. These results show the metastatic inhibition of cervical cancer by luteolin and quercetin through reducing UBE2S expression, and provide a functional role for UBE2S in the motility of cervical cancer. UBE2S could be a potential therapeutic target in cervical cancer.

Resveratrol suppresses myofibroblast activity of human buccal mucosal fibroblasts through the epigenetic inhibition of ZEB1 expression.

Oral submucous fibrosis (OSF) is a precancerous condition of the oral mucosa without specific therapeutic drugs. We previously demonstrated that the zinc finger E-box binding homeobox 1 (ZEB1) plays a pathogenic role in the induction of the myofibroblast activity of buccal mucosal fibroblasts (BMFs) and contributes to the pathogenesis of OSF. Resveratrol is a natural polyphenolic flavonoid with anti-fibrosis activity in various tissues and has the capability to inhibit ZEB1 in oral cancer cells. We examined the effect of resveratrol on the myofibroblast activity of human primary fibrotic BMFs (fBMFs) derived from OSF tissues. With the collagen contraction assay, resveratrol displayed anti-myofibroblast activity in three fBMF lines. Resveratrol also inhibited the expression of fibrogenic genes at the mRNA and protein levels in a dose- and time-dependent manner. The downregulation of ZEB1 in fBMFs by resveratrol was mediated by epigenetic mechanisms, such as the upregulated expression of miR-200c and the enhancer of zeste homolog 2 (EZH2), as well as the trimethylated lysine 27 of histone H3 (H3K27me3). Resveratrol also increased the binding of H3K27me3 to the ZEB1 promoter. The knockdown of EZH2 in fBMFs caused the upregulation of ZEB1 and suppressed the inhibitory effect of resveratrol. Furthermore, the reversed expression pattern between EZH2 and ZEB1 was observed in 6/8 OSF tissues with twofold upregulation of ZEB1 expression compared with the adjacent normal mucosa. In conclusion, our data suggest that resveratrol epigenetically inhibits ZEB1 expression to suppress the myofibroblast activity of fBMFs and may serve as a dietary supplement for OSF patients.

Effect of Grape Seed Proanthocyanidin-Gelatin Colloidal Complexes on Stability and in Vitro Digestion of Fish Oil Emulsions.

The colloidal complexes composed of grape seed proanthocyanidin (GSP) and gelatin (GLT), as natural antioxidants to improve stability and inhibit lipid oxidation in menhaden fish oil emulsions, were evaluated. The interactions between GSP and GLT, and the chemical structures of GSP/GLT self-assembled colloidal complexes, were characterized by isothermal titration calorimetry (ITC), circular dichroism (CD), and Fourier transform infrared spectroscopic (FTIR) studies. Fish oil was emulsified with GLT to obtain an oil-in-water (o/w) emulsion. After formation of the emulsion, GLT was fixed by GSP to obtain the GSP/GLT colloidal complexes stabilized fish oil emulsion. Menhaden oil emulsified by GSP/GLT(0.4 wt %) colloidal complexes yielded an emulsion with smaller particles and higher emulsion stability as compared to its GLT emulsified counterpart. The GSP/GLT colloidal complexes inhibited the lipid oxidation in fish oil emulsions more effectively than free GLT because the emulsified fish oil was surrounded by the antioxidant GSP/GLT colloidal complexes. The digestion rate of the fish oil emulsified with the GSP/GLT colloidal complexes was reduced as compared to that emulsified with free GLT. The extent of free fatty acids released from the GSP/GLT complexes stabilized fish oil emulsions was 63.3% under simulated digestion condition, indicating that the fish oil emulsion was considerably hydrolyzed with lipase.

Delivery of berberine using chitosan/fucoidan-taurine conjugate nanoparticles for treatment of defective intestinal epithelial tight junction barrier.

Bacterial-derived lipopolysaccharides (LPS) can cause defective intestinal barrier function and play an important role in the development of inflammatory bowel disease. In this study, a nanocarrier based on chitosan and fucoidan was developed for oral delivery of berberine (Ber). A sulfonated fucoidan, fucoidan-taurine (FD-Tau) conjugate, was synthesized and characterized by Fourier transform infrared (FTIR) spectroscopy. The FD-Tau conjugate was self-assembled with berberine and chitosan (CS) to form Ber-loaded CS/FD-Tau complex nanoparticles with high drug loading efficiency. Berberine release from the nanoparticles had fast release in simulated intestinal fluid (SIF, pH 7.4), while the release was slow in simulated gastric fluid (SGF, pH 2.0). The effect of the berberine-loaded nanoparticles in protecting intestinal tight-junction barrier function against nitric oxide and inflammatory cytokines released from LPS-stimulated macrophage was evaluated by determining the transepithelial electrical resistance (TEER) and paracellular permeability of a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran) in a Caco-2 cells/RAW264.7 cells co-culture system. Inhibition of redistribution of tight junction ZO-1 protein by the nanoparticles was visualized using confocal laser scanning microscopy (CLSM). The results suggest that the nanoparticles may be useful for local delivery of berberine to ameliorate LPS-induced intestinal epithelia tight junction disruption, and that the released berberine can restore barrier function in inflammatory and injured intestinal epithelial.

MicroRNA-302b-inhibited E2F3 transcription factor is related to all trans retinoic acid-induced glioma cell apoptosis.

All-trans retinoic acid (ATRA), a derivative of retinoid, is involved in the onset of differentiation and apoptosis in a wide variety of normal and cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression. Several miRNAs were identified to participate in ATRA-mediated cell differentiation. However, no studies have demonstrated whether miRNA can enhance ATRA cytotoxicity, thereby resulting in cell apoptosis. This study investigated the effects of ATRA-mediated miRNA expression in activating apoptotic pathways in glioblastoma. First, we found that high-dose ATRA treatment significantly reduced cell viability, caspase-dependent apoptosis, endoplasmic reticular (ER) stress activation, and intracellular reactive oxygen species accumulation. From microarray data, miR-302b was analyzed as a putative downstream regulator upon ATRA treatment. Furthermore, we found that ATRA up-regulated miR-302b expression in a dose- and time-dependent manner through retinoic acid receptor α-mediated pathway. Overexpression and knockdown of miR-302b significantly influenced ATRA-mediated cytotoxicity. E2F3, an important transcriptional regulator of glioma proliferation, was validated to be a direct target gene of miR-302b. The miR-302b-reduced E2F3 levels were also identified to be associated with ATRA-mediated glioma cell death. These results emphasize that an ATRA-mediated miR-302b network may provide novel therapeutic strategies for glioblastoma therapy. We propose that high-dose all-trans retinoic acid (ATRA) treatment, a derivative of retinoid, significantly induces glioblastoma cell apoptosis via caspase-dependent apoptosis, endoplasmic reticular (ER) stress, and intracellular reactive oxygen species (ROS) accumulation. The miR-302b overexpression enhanced by ATRA-mediated retinoic acid receptor (RAR)α pathway was also identified. The E2F3 repression, a novel target gene of miR-302b, was involved in ATRA-induced glioblastoma cell cytotoxicity.

Wogonin but not Nor-wogonin inhibits lipopolysaccharide and lipoteichoic acid-induced iNOS gene expression and NO production in macrophages.

Wogonin (Wog; 5,7-dihydroxy-8-methoxy flavone) has been shown to effectively inhibit lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide production in our previous study. In the present study, we found that Nor-wogonin (N-Wog; 5,7,8-trihydroxyl flavone), a structural analogue of Wog with an OH substitution at C8, performed different effect on LPS- or lipoteichoic acid (LTA)-induced iNOS gene expression and nitric oxide (NO) production in macrophages. Wog, but not N-Wog, significantly inhibits LPS- or LTA-induced NO production through suppressing iNOS gene expression at both protein and mRNA without affecting NO donor sodium nitroprusside-induced NO production, NOS enzyme activity, and cells viability. Activation of JNKs (not ERKs) via phosphorylation induction, and an increase in c-Jun (not c-Fos) protein expression were involved in LPS- and LTA-treated RAW264.7 cells, and those events were blocked by Wog, but not N-Wog, addition. Furthermore, 5,7-diOH flavone, but not 5-OH flavone, 7-OH flavone, 5-OH-7-OCH(3) flavone, significantly inhibits LPS-induced iNOS protein expression and NO production, and 7,8-diOCH(3) flavone performs more effective inhibitory activity on LPS-induced NO production and iNOS protein expression than 7-OCH(3)-8-OH flavone. These data suggest that OHs at both C5 and C7 are essential for NO inhibition of flavonoids, and OCH(3) at C8 may contribute to this activity, and suppression of JNKs-c-Jun activation is involved.