Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>A tannic acid-inducible and mycoviral-regulated laccase3 (<it>lac</it>3) from the chestnut blight fungus <it>Cryphonectria parasitica </it>has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast <it>Saccharomyces cerevisiae</it>.</p> <p>Results</p> <p>Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the <it>lac</it>3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned <it>lac</it>3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura^-media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of <it>lac</it>3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura^-media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the <it>lac</it>3 gene resulted in a reduced cell number of the <it>lac</it>3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without <it>lac</it>3 induction did not affect the growth of transformants.</p> <p>Conclusions</p> <p>The results suggest that expression of the <it>lac</it>3 gene has an inhibitory effect on the growth of transformed <it>S. cerevisiae </it>and that the controlled expression of <it>lac</it>3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.</p

Human Neural Stem Cells Genetically Modified to Overexpress Akt1 Provide Neuroprotection and Functional Improvement in Mouse Stroke Model

In a previous study, we have shown that human neural stem cells (hNSCs) transplanted in brain of mouse intracerebral hemorrhage (ICH) stroke model selectively migrate to the ICH lesion and induce behavioral recovery. However, low survival rate of grafted hNSCs in the brain precludes long-term therapeutic effect. We hypothesized that hNSCs overexpressing Akt1 transplanted into the lesion site could provide long-term improved survival of hNSCs, and behavioral recovery in mouse ICH model. F3 hNSC was genetically modified with a mouse Akt1 gene using a retroviral vector. F3 hNSCs expressing Akt1 were found to be highly resistant to H2O2-induced cytotoxicity in vitro. Following transplantation in ICH mouse brain, F3.Akt1 hNSCs induced behavioral improvement and significantly increased cell survival (50–100% increase) at 2 and 8 weeks post-transplantation as compared to parental F3 hNSCs. Brain transplantation of hNSCs overexpressing Akt1 in ICH animals provided functional recovery, and survival and differentiation of grafted hNSCs. These results indicate that the F3.Akt1 human NSCs should be a great value as a cellular source for the cellular therapy in animal models of human neurological disorders including ICH

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust <it>in vitro </it>RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.</p> <p>Results</p> <p>To characterize the biochemical properties of JEV RdRp, we expressed in <it>Escherichia coli </it>and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U₈₁, at the two nucleotides upstream of the 3'-end of the template.</p> <p>Conclusion</p> <p>As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the <it>in vitro </it>reconstitution of viral RNA replicase complex, we for the first time established an <it>in vitro </it>JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.</p

Effectiveness of vaccination and quarantine policies to curb the spread of COVID-19

A pandemic, the worldwide spread of a disease, can threaten human beings from the social as well as biological perspectives and paralyze existing living habits. To stave off the more devastating disaster and return to a normal life, people make tremendous efforts at multiscale levels from individual to worldwide: paying attention to hand hygiene, developing social policies such as wearing masks, social distancing, quarantine, and inventing vaccines and remedy. Regarding the current severe pandemic, namely the coronavirus disease 2019, we explore the spreading-suppression effect when adopting the aforementioned efforts. Especially the quarantine and vaccination are considered since they are representative primary treatments for block spreading and prevention at the government level. We establish a compartment model consisting of susceptible (S), vaccination (V), exposed (E), infected (I), quarantined (Q), and recovered (R) compartments, called SVEIQR model. We look into the infected cases in Seoul and consider three kinds of vaccines, Pfizer, Moderna, and AstraZeneca. The values of the relevant parameters are obtained from empirical data from Seoul and clinical data for vaccines and estimated by Bayesian inference. After confirming that our SVEIQR model is plausible, we test the various scenarios by adjusting the associated parameters with the quarantine and vaccination policies around the current values. The quantitative result obtained from our model could suggest a guideline for policy making on effective vaccination and social policies.Comment: 8 pages, 5 figure

Consensus of Corporate E-Learning System Stakeholders Regarding the Satisfaction of End-Users

The purpose of this study is to call attention to the consensus of stakeholders of corporate e-Learning system regarding success. We identified the critical success factors (contents, technical features, management, and organizational support) as major components of corporate eLearning systems and questioned whether stakeholders’ consensus on the importance of these components facilitates the implementation of these components to achieve good quality or well. We also questioned whether the influence of these components on user satisfaction could be moderated by contextual factors. Based on empirical testing of 18 eLearning user companies, we verified that the consensus of stakeholders regarding the importance of content, technological features, and organizational support has a positive influence on the perceived quality of these factors in their e-Learning systems, which in turn is positively related to user satisfaction. The learning subjects and learning style did significantly moderate the influences of these perceived qualities on user satisfaction

Extraskeletal chondroma of the fallopian tube.

Extraskeletal chondroma can occur in the hands, feet, head and neck. This tumor usually presents as a small solitary nodule. The histogenesis of the tumor is controversial, but some have suggested a metaplastic origin. Chondroma of the fallopian tube is very rare. There is only one report in English literature. The origin of this tumor can be subcoelomic mesenchyme of the tubal serosa or mesenchyme of the myosalpinx. We describe a case of chondroma arising from the serosal surface of the fallopian tube with a review of literature. A 30-yr-old woman visited hospital due to left adnexal mass. On operating finding, 2 x 3 cm sized nodular mass was noted on the left tubal serosal area. The excised mass showed multilobulated appearance covered with thin fibrous membrane. The cut surface was solid, grayish yellow, and myxoid with a focal gelatinous area. The microscopic finding showed islands and elongated lobules of mature benign cartilage without cytologic atypia

Carnosol induces apoptotic cell death through ROS-dependent inactivation of STAT3 in human melanoma G361 cells

Melanoma is the leading cause of skin cancer deaths, and the poor prognosis of metastatic melanoma has made needs for a novel pharmacological treatment or efficient intervention. Carnosol, a major polyphenolic compound from Rosmarinus officinalis, has a wide range of biological activities including anti-cancer effect. However, the underlying molecular mechanisms of its anti-cancer effect remain poorly understood in malignant human melanoma cells. In the present study, we investigate the apoptotic effect and the underlying anti-cancer mechanisms of carnosol. Our results revealed that carnosol strongly induced apoptosis against human melanoma G361 cells in a dose- and time-dependent manner, and caused dramatical elevation in cellular reactive oxygen species (ROS) level during apoptosis. In mechanistic studies, carnosol treatment decreased protein level of anti-apoptotic B‑cell lymphoma 2 (Bcl-2) and B cell lymphoma-extra large (Bcl-xL), however, increased level of pro-apoptotic Bcl-2-associated X protein (Bax) protein. Moreover, carnosol escalated cellular level of p53, which was accompanied by a decline of mouse double minute 2 homolog (MDM2) level. Also, carnosol inhibited activation of Src and signal transducer and activator of transcription 3 (STAT3), therefore down-regulated STAT3-dependent gene expression, such as D-series cyclin and survivin. These changes by carnosol were attenuated by pre-treatment of N-acetyl cysteine, and abolished progression of carnosol-induced apoptosis. In conclusion, carnosol induced apoptosis in human melanoma G361 cells through ROS generation and inhibition of STAT3-mediated pathway. Our results provide molecular bases of carnosol-induced apoptosis, and suggest a novel candidate for human melanoma treatment.This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1A02050495, J.-S. Choi) and by the Ministry of Science, ICT and Future Planning 2017R1A2B4009831, K.- S. Chun)