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

Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway

AbstractThe biologically active factors known as adipocytokines are secreted primarily by adipose tissues and can act as modulators of angiogenesis. Visfatin, an adipocytokine that has recently been reported to have angiogenic properties, is upregulated in diabetes, cancer, and inflammatory diseases. Because maintenance of an angiogenic balance is critically important in the management of these diseases, understanding the molecular mechanism by which visfatin promotes angiogenesis is very important. In this report, we describe our findings demonstrating that visfatin stimulates the mammalian target of the rapamycin (mTOR) pathway, which plays important roles in angiogenesis. Visfatin induced the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) in human endothelial cells. Inhibition of the mTOR pathway by rapamycin eliminated the angiogenic and proliferative effects of visfatin. The visfatin-induced increase in VEGF expression was also eliminated by RNA interference-mediated knockdown of the 70-kDa ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR. Visfatin inactivated glycogen synthase kinase 3β (GSK3β) by phosphorylating it at Ser-9, leading to the nuclear translocation of β-catenin. Both rapamycin co-treatment and p70S6K knockdown inhibited visfatin-induced GSK3β phosphorylation at Ser-9 and nuclear translocation of β-catenin. Taken together, these results indicate that mTOR signaling is involved in visfatin-induced angiogenesis, and that this signaling leads to visfatin-induced VEGF expression and nuclear translocation of β-catenin

Geriatric Medicine in South Korea: A Stagnant Reality amidst an Aging Population

In the face of an ever-increasing wave of an aging population, this paper provides an update on the current status of geriatric medicine in Korea, comparing it with global initiatives and suggesting future directions. Older adults require a multifaceted approach, addressing not only comorbidity management but also unmet complex medical needs, nutrition, and exercise to prevent functional decline. In this regard, the World Health Organization's Integrated Care for Older People guidelines underscore the importance of patient-centered primary care in preventing a decline in intrinsic capacity. Despite these societal needs and the ongoing aging process, the healthcare system in Korea has yet to show significant movement or a shift toward geriatric medicine, further complicated by the absence of a primary care system. We further explore global efforts in establishing age-integrative patient-centered medical systems in Singapore, Australia, Canada, the United Kingdom, and Japan. Additionally, we review the unmet needs and social issues that Korean society is currently facing, and local efforts by both government and a private tertiary hospital in Korea. In conclusion, considering the current situation, we propose that the framework of geriatric medicine should form the foundation of the future healthcare system

Combined Effects of Surface Morphology and Mechanical Straining Magnitudes on the Differentiation of Mesenchymal Stem Cells without Using Biochemical Reagents

Existing studies examining the control of mesenchymal stem cell (MSC) differentiation into desired cell types have used a variety of biochemical reagents such as growth factors despite possible side effects. Recently, the roles of biomimetic microphysical environments have drawn much attention in this field. We studied MSC differentiation and changes in gene expression in relation to osteoblast-like cell and smooth muscle-like cell type resulting from various microphysical environments, including differing magnitudes of tensile strain and substrate geometries for 8 days. In addition, we also investigated the residual effects of those selected microphysical environment factors on the differentiation by ceasing those factors for 3 days. The results of this study showed the effects of the strain magnitudes and surface geometries. However, the genes which are related to the same cell type showed different responses depending on the changes in strain magnitude and surface geometry. Also, different responses were observed three days after the straining was stopped. These data confirm that controlling microenvironments so that they mimic those in vivo contributes to the differentiation of MSCs into specific cell types. And duration of straining engagement was also found to play important roles along with surface geometry

Piceatannol, Natural Polyphenolic Stilbene, Inhibits Adipogenesis via Modulation of Mitotic Clonal Expansion and Insulin Receptor-dependent Insulin Signaling in Early Phase of Differentiation

Piceatannol, a natural stilbene, is an analog and a metabolite of resveratrol. Despite a well documented health benefit of resveratrol in intervention of the development of obesity, the role of piceatannol in the development of adipose tissue and related diseases is unknown. Here, we sought to determine the function of piceatannol in adipogenesis and elucidate the underlying mechanism. We show that piceatannol inhibits adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner at noncytotoxic concentrations. This anti-adipogenic property of piceatannol was largely limited to the early event of adipogenesis. In the early phase of adipogenesis, piceatannol-treated preadipocytes displayed a delayed cell cycle entry into G2/M phase at 24 h after initiation of adipogenesis. Furthermore, the piceatannol-suppressed mitotic clonal expansion was accompanied by reduced activation of the insulin-signaling pathway. Piceatannol dose-dependently inhibited differentiation mixture-induced phosphorylation of insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/Akt pathway in the early phase of adipogenesis. Moreover, we showed that piceatannol is an inhibitor of IR kinase activity and phosphatidylinositol 3-kinase (PI3K). Our kinetics study of IR further identified a Km value for ATP of 57.8 μm and a Ki value for piceatannol of 28.9 μm. We also showed that piceatannol directly binds to IR and inhibits IR kinase activity in a mixed noncompetitive manner to ATP, through which piceatannol appears to inhibit adipogenesis. Taken together, our study reveals an anti-adipogenic function of piceatannol and highlights IR and its downstream insulin signaling as novel targets for piceatannol in the early phase of adipogenesis

Activation of spleen tyrosine kinase is required for TNF-α-induced endothelin-1 upregulation in human aortic endothelial cells

AbstractEndothelin-1 (ET-1) promotes atherosclerosis. We tested whether spleen tyrosine kinase (Syk) mediates tumor necrosis factor-α (TNF-α)-induced ET-1 upregulation in human aortic endothelial cells (HAECs) and sought to identify the signal pathways involved. TNF-α-induced reactive oxygen species (ROS) activated Syk and phosphatidylinositol 3-kinase (PI3K), which was required for the activation of AP-1 and subsequent ET-1 gene transcription. ROS mediated c-Jun NH2-terminal kinase (JNK) is also required for AP-1 activation, but Syk and PI3K regulated AP-1 activation independently of JNK. Through regulation of ET-1 production, Syk could be implicated in atherosclerosis

APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF

Pancreatic cancer is the worst exocrine gastrointestinal cancer leading to the highest mortality. Recent studies reported that aberrant expression of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is involved in uncontrolled cell growth. However, the molecular mechanism of APE1 biological role remains unrevealed in pancreatic cancer progression. Here, we demonstrate that APE1 accelerates pancreatic cancer cell proliferation through glial cell line-derived neurotrophic factor (GDNF)/glial factor receptor α1 (GFRα1)/Src/ERK axis-cascade signaling. The proliferation of endogenous APE1 expressed-MIA PaCa-2, a human pancreatic carcinoma cell line, was increased by treatment with GDNF, a ligand of GFRα1. Either of downregulated APE1 or GFRα1 expression using small interference RNA (siRNA) inhibited GDNF-induced cancer cell proliferation. The MEK-1 inhibitor PD98059 decreased GDNF-induced MIA PaCa-2 cell proliferation. Src inactivation by either its siRNA or Src inhibitor decreased ERK-phosphorylation in response to GDNF in MIA PaCa-2 cells. Overexpression of GFRα1 in APE1-deficient MIA PaCa-2 cells activated the phosphorylation of Src and ERK. The expression of both APE1 and GFRα1 was gradually increased as progressing pancreatic cancer grades. Our results highlight a critical role for APE1 in GDNF-induced pancreatic cancer cell proliferation through APE1/GFRα1/Src/ERK axis-cascade signaling and provide evidence for future potential therapeutic drug targets for the treatment of pancreatic cancer

Does Diabetes Mellitus Influence Standardized Uptake Values of Fluorodeoxyglucose Positron Emission Tomography in Colorectal Cancer?

Background/AimsHyperglycemia is associated with decreased 2-18[F]fluoro-2-deoxy-D-glucose (FDG) uptake by tumors assessed by positron emission tomography (PET). In this retrospective study we investigated a comparison of standardized uptake values (SUVs) in patients with primary colorectal cancers who either had diabetes mellitus (DM) or were otherwise healthy.MethodsThe medical records of 397 patients who were diagnosed with colorectal cancer and underwent PET-CT between January 2006 and December 2012 were analyzed. Eighty patients with DM and 317 patients without DM were included. Clinical characteristics were reviewed and maximal standardized uptake values (SUVmax) were calculated in the primary colorectal lesions.ResultsThere was no significant difference between tumor SUVmax in DM patients (10.60±5.78) and those without DM (10.92±5.44). In addition, no significant difference was detected between tumor SUVmax in DM patients with glycated hemoglobin (HbA1c) levels <8% (10.34±5.17) and those with HbA1c levels ≥8% (10.61±7.27). The maximum size of the primary colorectal tumor was associated with SUVmax in a linear regression analysis.ConclusionThe results of this study showed that DM did not influence FDG uptake values in colorectal cancer patients regardless of glucose levels