Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2) gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7) cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO) QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth

Efficient transgene expression system using a cumate-inducible promoter and Cre-loxP recombination in avian cells

Objective Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression

Intraventricular Hemorrhage Caused by Lateral Ventricular Meningioma: A Case Report

Meningiomas causing intracranial hemorrhage are rare, and hemorrhage from a lateral ventricular meningioma seems to be even rarer. We report a case of trigonal meningioma in a 43-year-old woman who presented with intraventricular hemorrhage, and describe the CT, MRI and angiographic findings

Influence of prohexadione-calcium, trinexapac-ethyl and hexaconazole on lodging characteristic and gibberellin biosynthesis of rice (Oryza sativa L.)

We investigated the influence of prohexadione-calcium (Pro-Ca), trinexapac-ethyl (TNE) and hexaconazole (HX) on lodging and gibberellin (GA) biosynthesis pathway of rice cultivar, Hwayeongbyeo. It was observed that these novel synthetic growth retardants suppressed lodging of rice under field conditions through blocking GA biosynthesis pathway. These growth retarding chemicals were applied at basic (20 uM) and elevated (40 uM) rates either 10 days before heading (10 DBH) or 5 days before heading (5 DBH). We found that Pro-Ca, TNE and their combined application (Pro-Ca + TNE) were most effective in decreasing rice length and lodging index, when applied at 10 DBH. Similarly, the endogenous bioactive GA1 contents of rice significantly declined with application of Pro-Ca, TNE and Pro-Ca + TNE, while they were less effected by basic and elevated rates of HX as compared to the control. The growth retardants were more effective in decreasing rice lodging and blocking GA biosynthesis when applied in elevated rates. The levels of the endogenous gibberellins in rice shoots were measured by GC/MS-SIM using 2H2-labeled gibberellins as internal standards. Effect of these synthetic chemicals on growth and GA inhibition were stronger initially but eroded rapidly under field conditions. It was thus concluded that Pro-Ca and TNE were most effective in reducing plant length and suppressing lodging of rice crop under field conditions, where lodging is a major constraint to higher productivity.Key words: Growth retardants, plant growth, gibberellin biosynthesis, lodging index, rice

Anti-inflammatory effects of enzymatic hydrolysates of velvet antler in RAW 264.7 cells in vitro and zebrafish model

Enzymatic hydrolysis has been successfully used for the extraction of numerous biologically active components from a wide variety of natural sources. In the present study, velvet antler was subjected to the extraction process using Alcalase protease. We analyzed bioactive components, such as uronic acid, sulfated-glycosaminoglycans (sulfated-GAGs), and sialic acid, present in the velvet antler Alcalase hydrolysate (VAAH) and assessed their anti-inflammatory effects in zebrafish as well as in vitro using cell lines. VAAH mainly contained uronic acid (78.22 mg/g) and sulfated-GAGs (50.47 mg/g), while the amount of sialic acid was negligible (5.55 mg/g). VAAH inhibited the production of nitric oxide (NO) by lipopolysaccharide (LPS)-induced cells in a dosedependent manner and the inhibitory effect of VAAH on NO production was higher than that of hot water extracts. VAAH treatment also reduced the expression of inflammatory mediators such as nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, we evaluated anti-inflammatory effects of VAAH using LPS-stimulated zebrafish. Treatment with LPS significantly increased cell death, NO, and reactive oxygen species (ROS) levels in zebrafish. Notably, VAAH significantly inhibited the extent of LPS-stimulated cell death and generation of NO and ROS in zebrafish. These results suggest that VAAH alleviated inflammation and cell death by inhibiting the generation of ROS induced by LPS treatment. Thus, VAAH could be used as a potential natural remedy with a trong anti-inflammatory effect. Taken together, we believe that based on our present results, enzymatic hydrolysis of velvet antler may be an effective process to make antler products acceptable as elements of health foods and nutraceutical components with increased biological activity

Preclinical evaluation of injectable sirolimus formulated with polymeric nanoparticle for cancer therapy

Nanoparticles are useful delivery vehicles for promising drug candidates that face obstacles for clinical applicability. Sirolimus, an inhibitor of mammalian target of rapamycin has gained attention for targeted anticancer therapy, but its clinical application has been limited by its poor solubility. This study was designed to enhance the feasibility of sirolimus for human cancer treatment. Polymeric nanoparticle (PNP)–sirolimus was developed as an injectable formulation and has been characterized by transmission electron microscopy and dynamic light scattering. Pharmacokinetic analysis revealed that PNP–sirolimus has prolonged circulation in the blood. In addition, PNP–sirolimus preserved the in vitro killing effect of free sirolimus against cancer cells, and intravenous administration displayed its potent in vivo anticancer efficacy in xenograft tumor mice. In addition, PNP–sirolimus enhanced the radiotherapeutic efficacy of sirolimus both in vitro and in vivo. Clinical application of PNP–sirolimus is a promising strategy for human cancer treatment

Photophysical Properties of Pheophorbide-a Derivatives and Their Photodynamic Therapeutic Effects on a Tumor Cell Line In Vitro

Pheophorbide-a derivatives have been reported to be potential photosensitizers for photodynamic therapy (PDT). In this study, photophysics of pheophorbide-a derivatives (PaDs) were investigated along with their photodynamic tumoricidal effect in vitro. PaDs were modified by changing the coil length and/or making the hydroxyl group (–OH) substitutions. Their photophysical properties were studied by steady-state and time-resolved spectroscopic methods. The photodynamic tumoricidal effect was evaluated in the mouse breast cancer cell line (EMT6). Lifetime and quantum yield of fluorescence and quantum yields of triplet state and singlet oxygen were studied to determine the dynamic energy flow. The coil length of the substituted alkyl group did not significantly affect the spectroscopic properties. However, the substitution with the hydroxyl group increased the quantum yields of the triplet state and the singlet oxygen due to the enhanced intersystem crossing. In order to check the application possibility as a photodynamic therapy agent, the PaDs with hydroxyl group were studied for the cellular affinity and the photodynamic tumoricidal effect of EMT6. The results showed that the cellular affinity and the photodynamic tumoricidal effect of PaDs with the hydroxyl group depended on the coil-length of the substituted alkyl group