m6Aexpress-enet: Predicting the regulatory expression m6A sites by an enet-regularization negative binomial regression model.

As the most abundant mRNA modification, m6A controls and influences many aspects of mRNA metabolism including the mRNA stability and degradation. However, the role of specific m6A sites in regulating gene expression still remains unclear. In additional, the multicollinearity problem caused by the correlation of methylation level of multiple m6A sites in each gene could influence the prediction performance. To address the above challenges, we propose an elastic-net regularized negative binomial regression model (called m6Aexpress-enet) to predict which m6A site could potentially regulate its gene expression. Comprehensive evaluations on simulated datasets demonstrate that m6Aexpress-enet could achieve the top prediction performance. Applying m6Aexpress-enet on real MeRIP-seq data from human lymphoblastoid cell lines, we have uncovered the complex regulatory pattern of predicted m6A sites and their unique enrichment pathway of the constructed co-methylation modules. m6Aexpress-enet proves itself as a powerful tool to enable biologists to discover the mechanism of m6A regulatory gene expression. Furthermore, the source code and the step-by-step implementation of m6Aexpress-enet is freely accessed at https://github.com/tengzhangs/m6Aexpress-enet.

Formulation of a Self-Nanoemulsifying Drug Delivery System of Buckwheat Flavonoids and Evaluation of Its Antimicrobial Activity.

This study was aimed at constructing a self-nanoemulsifying drug delivery system of buckwheat flavonoids and evaluating its antimicrobial activity. The construction of the nanoemulsion followed a pseudo-ternary phase diagram, and its particle properties (particle size, zeta potential, and surface morphology) and physicochemical parameters (turbidity, surface tension, pH value, conductivity, encapsulation efficiency, and stability) were evaluated. The antimicrobial potential of buckwheat flavonoids nanoemulsion was determined against Staphylococcus aureus, Escherichia coli, and Candida albicans and compared to the buckwheat flavonoids suspension. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) exhibited that the antimicrobial activity of the nanoemulsions and suspension increased while enhancing the drug concentration, and the antimicrobial activity of nanoemulsion was significantly higher than that of the suspension against those three bacteria. Agar disc diffusion test demonstrated that the inhibition zone diameter of the suspension was about 50% of the nanoemulsion against three bacteria. The time killing assay indicated that the IC50 of the nanoemulsion was significantly lower than that of the suspension. These results indicate that nanoemulsion is a promising drug delivery system, which can improve the antimicrobial activity of buckwheat flavonoids.

Function of Krüppel­like factor 2 in the shear stress­induced cell differentiation of endothelial progenitor cells to endothelial cells.

The present study aimed to evaluate the effects of Krüppel­like factor 2 (KLF2) on the differentiation of endothelial progenitor cells (EPCs) to endothelial cells (ECs) induced by shear stress, and to investigate the corresponding mechanisms. Cultured rat late EPCs were exposed to shear stress (12 dyn/cm2) for different lengths of time. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was used to measure the initial KLF2 mRNA levels in each group. Subsequently, the EPCs were treated with anti­integrin ß1 or ß3 antibodies to block integrin ß1 and ß3, respectively, or cytochalasin D to destroy F­actin, and the subsequent expression levels of KLF2 in EPCs were measured. Then, KLF2 small interfering RNAs (siRNAs) were transfected into EPCs, and RT­qPCR was used to measure the mRNA expression level of KLF2. Additionally, flow cytometry was applied to evaluate the protein levels of cluster of differentiation 31 (CD31) and the von Willebrand factor (vWF), and the regulatory effects of KLF2 in the promoter region of vWF were determined via a luciferase assay. High shear stress upregulated KLF2 expression, while blocking integrin ß1/ß3 or destroying F­actin resulted in a corresponding decrease in KLF2 expression. Downregulation of KLF2 expression by siKLF2 inhibited the differentiation of EPCs to ECs under shear stress conditions, while the expression of EC­specific markers decreased, including CD31 and vWF. Various lengths of the vWF promoter region induced vWF expression, and EPCs co­transfected with KLF2 significantly increased the vWF expression levels compared with the group treated with vWF alone (P<0.01). In conclusion, shear stress may upregulate KLF2 expression, which may be associated with the integrin­actin cytoskeleton system. Most importantly, the shear stress­induced differentiation of EPCs may be mediated by KLF2.

[Effects of endothelial progenitor cells on proliferation and biological function of hepatic stellate cells under shear stress].

OBJECTIVE: To investigate the effects of endothelial progenitor cells (EPCs) under shear stress on the biological function such as proliferation, adhesion, migration, apoptosis and expression of α-smooth muscle actin (α-SMA), collagen-I and collagen-Ⅲ of hepatic stellate cells (HSCs). METHODS: HSCs and EPCs were inoculated into the upper and lower layers of the co-culture chamber respectively and co-incubated for 24 hours. Then, 12 dyne/cm2 shear stress was applied to EPCs cells for another 24 hours. After that, proliferation, adhesion, migration and apoptosis of HSCs were detected by cell counting kit-8 (CCK-8) kit, cell adherent assay, Boyden cell migration assay and flow cytometry respectively. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of alpha -SMA, collagen I and collagen-Ⅲ in HSCs. RESULTS: Under shear stress, EPCs ecological niche could obviously inhibit the proliferation, adhesion and migration of HSCs, promote the apoptosis of HSCs, and down-regulate the mRNA and protein expression of collagen-I, collagen-Ⅲ in HSC cells. CONCLUSIONS: Under shear stress, EPCs ecological niche could inhibit the fibrosis development of HSCs to a certain extent.

A scheme for a single molecule phase-shift gate in a solid matrix.

We propose a feasible scheme to implement a phase-shift gate ((1 0) (0 eiγ)) based on a two-state single molecule in a solid matrix, where γ is a geometric phase controlled through a fast on-resonant laser field and a slow off-resonant radio-frequency field. In our scheme, a non-Hermitian quantum model is employed to characterize the single molecule in a solid matrix including the spontaneous decay effect. By the coupling between the radio-frequency field and the two-state permanent dipole difference resulting from the solid matrix, the spontaneous decay fatal to the preservation of geometric phase can be effectively suppressed for a considerably long waiting time.

[Effects of inward rectifier potassium channel blockers on EPCs function].

OBJECTIVE: To investigate the effects of inward rectifier potassium channel blockers (BaCl2, CsCl) on the functions of endothelial progenitor cells (EPCs). METHODS: Density gradient centrifugation-isolated rat hone marrow mononuclear cells were cultured in vitro. EPCs were harvested and seeded on six culture dish when cells grew to 3-5 passages. Before testing the EPCs were synchronized with M199, which contain 2% fetal calf serum. In the end, EPCs were treated with different intervention. The experiment mainly included two parts: (1) BaCl2 (100 micromol/L) and free BaC2 of Tyrodes solution; (2) CsCl (1 mmol/L) and control. Cell pretreated with blockers above mentioned for 12 h, then the gene expression of stromal cell-derived factor-1 (SDF-1), epoprotenol (PGI2) were assessed, beyond that the ability of adhesion, migration were assayed with different tests. In addition, the medium was collected when EPCs were treated for 3 days. The levels of SDF-1 were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Going even further, EPCs were treated with the signal pathway blockers in advance, after repeat the above steps, in order to analyze the change of SDF-1 and then discuss its mechanism. RESULTS: Compared with control group, BaCl2, CsCl could increase EPC adhesion and migration to same extent. Moreover, the gene expression of SDF-1, PGI2 was significantly up-regulated and the production of SDF-1 increased evidently. Furthermore, the mechanism of SDF-1 secretion increasing mainly was associated with eNOS signaling pathways. CONCLUSION: Ba2+ and Cs+ play important roles in increasing EPCs functions, such as adhesion, migration and secretion.

Protective effects of the active fraction from the tuber of Scirpus yagara in mouse endotoxin shock model.

ETHNOPHARMACOLOGICAL RELEVANCE: Scirpus yagara Ohwi is a perennial, aquatic plant, whose dry tubers have long been used as Traditional Chinese Medicine (TCM) "Sanleng" for the treatment of postpartum abdominal pain, hyperemesis gravidarum, amenorrhea, dyspepsia and several inflammatory related diseases. Although it is known to have anti-inflammatory activities, its mechanism of action on lipopolysaccharide (LPS)-induced inflammation has not yet been identified in detail.This study was designed to investigate the anti-inflammatory activity of the active fraction (AF) from the tuber of Scirpusyagara both in vitro and in vivo. MATERIALS AND METHODS: RAW264.7 macrophage was incubated for 16h with 1µg/ml of LPS in absence or presence of AF (0, 10, 50 and 100µg/ml) and the secretions of tumor necrosis-alpha (TNF-α) and interleukin-6 (IL-6) in the medium were determined by using an enzyme-linked immunosorbent assay (ELISA). In the in vivo study, mice were orally administrated with AF (50 and 300mg/kg) for three days consecutively. 1h after the last AF administration, the mice were intraperitoneally injected with LPS (15mg/kg), and the life span of LPS-challenged mice were determined. Furthermore, the levels of pro-inflammatory cytokines TNF-α and IL-6 in the serum, lung and liver were measured using ELISA kit, and histological change in lungs was examined by light microscopy. Additionally, the components of AF were analyzed by high performance liquid chromatography (HPLC) using a C18 column. RESULTS: AF significantly decreased TNF-α and IL-6 production induced by LPS in RAW264.7 macrophage. In LPS-induced mouse endotoxin shock model, AF pre-treatment significantly improved the survival rate of mice. And LPS-induced increases of pro-inflammatory cytokines TNF-α and IL-6 in the serum, lung and liver were markedly suppressed by AF. Moreover, the histopathological examination indicated that AF could significantly attenuate lung tissues injury in endotoxemic mice. In addition, eight compounds (protocatechuic acid, vanillic acid, p-coumaric acid, ferulic acid, methyl-3,6-dihydroxy-2-[2-(2-hydroxyphenyl)-ethynyl] benzoate, sciryagarol I, sparstolonin B, SanLeng diphenyllactone) of AF were quantified by HPLC analysis. CONCLUSIONS: These results suggested that AF protected mice against LPS-induced lethality by inhibiting the production of multiple cytokines and organ dysfunction. Thus AF may prove beneficial in the prevention and treatment of endotoxin shock.

Unification of the family of Garrison-Wright's phases.

Inspired by Garrison and Wight's seminal work on complex-valued geometric phases, we generalize the concept of Pancharatnam's "in-phase" in interferometry and further develop a theoretical framework for unification of the abelian geometric phases for a biorthogonal quantum system modeled by a parameterized or time-dependent nonhermitian hamiltonian with a finite and nondegenerate instantaneous spectrum, that is, the family of Garrison-Wright's phases, which will no longer be confined in the adiabatic and nonadiabatic cyclic cases. Besides, we employ a typical example, Bethe-Lamb model, to illustrate how to apply our theory to obtain an explicit result for the Garrison-Wright's noncyclic geometric phase, and also to present its potential applications in quantum computation and information.

[Effects of extracellular matrix on biological characteristics of late endothelial progenitor cells].

The present study was designed to investigate the effects of various extracellular matrix (ECM) proteins on the biological characteristics of late endothelial progenitor cells (EPCs). Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in complete M199 medium, which contained 15% fetal calf serum, 10 µg/L vascular endothelial growth factor (VEGF) and 5 µg/L basic fibroblast growth factor (bFGF). EPCs were plated on substrates containing fibronectin (Fn), laminin (Ln) or rat tail tendon collagen (Col), and the corresponding cells were defined as Fn, Ln and Col groups. The 3rd generation EPCs, namely late EPCs, were harvested. The proliferation, adhesion, migration and the ability of forming tubes were assayed using CCK-8, adhesion test, wound healing assay and Matrigel, respectively. The mRNA expressions of endothelial cell differentiation markers, vWF and CD31, were analyzed by real time RT-PCR. The apoptosis was assayed by flow cytometry (FCM). The results showed that cell proliferation ability of Fn and Col groups were higher than that of Ln group; Fn group showed increased adhesion compared to Col and Ln groups (P < 0.01); The migration ability of Fn and Col groups were higher than that of Ln group. Moreover, Fn group showed increased tube formation abilities compared to Col and Ln groups (P < 0.05). Although 24-hour free-serum-induced apoptosis in Ln group was the highest, there was no difference of auto-apoptosis among the three groups. Furthermore, the mRNA expressions of vWF and CD31 exhibited no difference among the three groups. These results suggest the ECM affects the biological functions of late EPCs, which would have a high probability of providing new directions that lead to the development of artificial heart and blood vessels.

[Anticancer activity of Crocodylus siamensis blood in vitro and in vivo].

OBJECTIVE: To study the anticancer effects of the blood of Crocodylus siamensis in vitro and in vivo. METHODS: The inhibitory effects of serum and plasma of Crocodylus siamensis on proliferation of HepG2, BGC823, HeLa and SKOV3 cell were measured by MTT assay. The mouse S180 tumor model was used to evaluate the anti-tumor effect in vivo. RESULTS: High dosage serum and plasma of Crocodylus siamensis could inhibit the proliferation of HepG2, BGC823, HeLa and SKOV3 cell. The tumor inhibitory rate of high dosage blood of Crocodylus siamensis on S180 tumor was up to 57.55%. CONCLUSION: The blood of breeding Crocodylus siamensis has anticancer activity.

Preparation and properties of rutin-hydrolyzing enzyme from tartary buckwheat seeds.

A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammonium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an apparent molecular weight of about 70kDa determined by SDS-PAGE, with an isoelectric point (pI) (determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incubated together with rutin at 37°C for 30min in the presence of 20% ethanol, and its Km value for rutin is 1.04×10(-3)M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and this is confirmed by (1)H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydrolyzing enzyme, providing a new enzymatic preparation method for quercetin.

[Preparation and identification of the rat antibody against recombinant buckwheat trypsin inhibitor].

AIM: To prepare the rat antibody against the recombinant buckwheat trypsin inhibitor. METHODS: The recombinant buckwheat trypsin inhibitor was expressed in E.coli. BL21 (DE3) and used as an immunogen to immunize the rat. The titer and specificity of the anti-BTI antibody from the rat were analyzed by ELISA, Western blot and immunohistochemistry, respectively. RESULTS: ELISA detection indicated the titer of the antiserum was about 1:128 000. Western blot analysis showed the antibody reacted specifically with rBTI. The immunohistochemistry analysis proved that rBTI was expressed in the cytoplast and nucleus of EC9706 cells. CONCLUSION: The rat antibody against rBTI has high titer and specificity, which is beneficial to further study on the molecular mechanisms in rBTI-induced apoptosis of tumor cells and also provide an important theory basis for further exploring the relationship between the structure and function of BTI.

Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat.

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.

[Effects of centipede extract on life span of drosophila].

OBJECTIVE: To evaluate effects of centipede on life span of drosophila and on the appearance of the filial generation. METHODS: Drosophila melanogaster (male and female) were cultured in tubes with centipede extraction at concentrations of 0, 0.2%, 1%, 2.5%, or 5% until all drosophila died. The appearance of the filial generation were observed. RESULTS: The life span, including average, longest and shortest life span was shortened in exposed groups of both sexes and significantly dose-dependent. The appearance of the filial generation showed normal. CONCLUSION: Centipede extracts accelerates the aging of drosophila and shortens their life span. The appearance of the filial generation is not affected. Clinic using centipede for a long period should be carefully.