Optimization of Hybrid Ink Formulation and IPL Sintering Process for Ink-Jet 3D Printing.

Ink-jet 3D printing technology facilitates the use of various materials of ink on each ink-jet head and simultaneous printing of multiple materials. It is suitable for manufacturing to process a complex multifunctional structure such as sensors and printed circuit boards. In this study, a complex structure of a SiO2 insulation layer and a conductive Cu layer was fabricated with photo-curable nano SiO2 ink and Intense Pulsed Light (IPL)-sinterable Cu nano ink using multi-material ink-jet 3D printing technology. A precise photo-cured SiO2 insulation layer was designed by optimizing the operating conditions and the ink rheological properties, and the resistance of the insulation layer was 2.43 × 1013 Ω·cm. On the photo-cured SiO2 insulation layer, a Cu conductive layer was printed by controlling droplet distance. The sintering of the IPL-sinterable nano Cu ink was performed using an IPL sintering process, and electrical and mechanical properties were confirmed according to the annealing temperature and applied voltage. Then, Cu conductive layer was annealed at 100 °C to remove the solvent, and IPL sintered at 700 V. The Cu conductive layer of the complex structure had an electrical property of 29 µΩ·cm and an adhesive property with SiO2 insulation layer of 5B.

Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis.

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors. In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model. We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4',6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways). Further, the anticancer effect in vivo was confirmed through a xenograft model. Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner. In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis. Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins. Additionally, shikonin was overexpressed in MAPK pathways. Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, pathologic changes were not observed in the liver and kidney of mice. Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.

Anti-cancer effect of GV1001 for prostate cancer: function as a ligand of GnRHR.

GV1001, a 16-amino acid fragment of the human telomerase reverse transcriptase catalytic subunit (hTERT), has been developed as an injectable formulation of cancer vaccine. Here, we revealed for the first time that GV1001 is a novel ligand for gonadotropin-releasing hormone receptor (GnRHR). The docking prediction for GV1001 against GnRHR showed high binding affinity. Binding of GV1001 to GnRHR stimulated the Gαs-coupled cAMP signaling pathway and antagonized Gαq-coupled Ca2+ release by leuprolide acetate (LA), a GnRHR agonist. Repeated injection of GV1001 attenuated both serum testosterone level and seminal vesicle weight via desensitization of hypothalamic-pituitary-gonadal (HPG) axis. We then tested whether GV1001 has an inhibitory effect on tumor growth of LNCaP cells, androgen receptor-positive human prostate cancer (PCa) cells. GV1001 significantly inhibited tumor growth and induced apoptosis in LNCaP-implanted xenografts. Interestingly, mRNA expressions of matrix metalloproteinase 2 and matrix metalloproteinase 9 were suppressed by GV1001, but not by LA. Moreover, GV1001 significantly inhibited the proliferation and migration of PCa cells and induced apoptosis in a concentration-dependent manner. Our findings suggest that GV1001 functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway, with anti-proliferative and anti-migratory effects on human PCa.

Immunomodulatory Potential of Weissella cibaria in Aged C57BL/6J Mice.

Aging is associated with distinct changes in immune cells and a decline in immune function, leading to increased susceptibility to infection and reduced responses to vaccination. Certain strains of lactic acid bacteria exert beneficial effects on the immune system. Previously, we reported that Weissella cibaria JW15 isolated from kimchi possesses immune stimulatory activity in vitro. In the present study, we further investigated whether oral administration of JW15 improves immune function in aged mice. Eighteen-month-old female mice were administered JW15 daily at low (JW15-L; 1 × 108 CFU/mouse) or high dosage (JW15-H; 1 × 109 CFU/mouse), or with Lactobacillus rhamnosus GG (LGG) using oral gavage. Twomonth- old female mice were included as healthy young mice. After 4 weeks, the mice were euthanized and immune profiles were analyzed using whole blood and spleen. In complete blood count analysis, the numbers of white and red blood cells were significantly increased in the JW15-L group compared with those in the old mouse (OM) control group. In addition, administration of either JW15 of LGG resulted in higher numbers of splenocytes in comparison with the OM group. Furthermore, proliferative potentials were higher in all probiotic groups than OM. Cytokines such as IFN-γ and IL-6 were secreted at higher levels in splenocytes isolated from JW15-fed mice than in OM control mice. Similarly, mRNA expression of various cytokines was altered in the JW15 groups. Collectively, these results suggest that JW15 supplementation induces immunomodulatory effects in aged mice and indicate JW15 as a potential probiotic strain to improve immune function in aged animals.

The Attenuation Phenotype of a Ribavirin-Resistant Porcine Reproductive and Respiratory Syndrome Virus Is Maintained during Sequential Passages in Pigs.

UNLABELLED: In a previous study, ribavirin-resistant porcine reproductive and respiratory syndrome virus (PRRSV) mutants (RVRp13 and RVRp22) were selected, and their resistance against random mutation was shown in cultured cells. In the present study, these ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability during three pig-to-pig passages in comparison with modified live virus (MLV) (Ingelvac PRRS MLV). Pigs challenged with RVRp22 had significantly lower (P< 0.05) viral loads in sera and tissues than pigs challenged with MLV or RVRp13 at the first passage, and the attenuated replication of RVRp22 was maintained until the third passage. Viral loads in sera and tissues dramatically increased in pigs challenged with MLV or RVRp13 during the second passage. Consistently, all five sequences associated with the attenuation of virulent PRRSV in RVRp13 and MLV quickly reverted to wild-type sequences during the passages, but two attenuation sequences were maintained in RVRp22 even after the third passage. In addition, RVRp22 showed a significantly lower (P< 0.001) mutation frequency in nsp2, which is one of the most variable regions in the PRRSV genome, than MLV. Nine unique mutations were found in open reading frames (ORFs) 1a, 2, and 6 in the RVRp22 genome based on full-length sequence comparisons with RVRp13, VR2332 (the parental virus of RVRp13 and RVRp22), and MLV. Based on these results, it was concluded that RVRp22 showed attenuated replication in pigs; further, because of the high genetic stability of RVRp22, its attenuated phenotype was stable even after three sequential passages in pigs. IMPORTANCE: PRRSV is a rapidly evolving RNA virus. MLV vaccines are widely used to control PRRS; however, there have been serious concerns regarding the use of MLV as a vaccine virus due to the rapid reversion to virulence during replication in pigs. As previously reported, ribavirin is an effective antiviral drug against many RNA viruses. Ribavirin-resistant mutants reemerged by escaping lethal mutagenesis when the treatment concentration was sublethal, and those mutants were genetically more stable than parental viruses. In a previous study, two ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22) were selected, and their higher genetic stability was shown in vitro Consequently, in the present study, both of the ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability in vivo RVRp22 was found to exhibit higher genetic and phenotypic stability than MLV, and nine unique mutations were identified in the RVRp22 genome based on a full-length sequence comparison with the RVRp13, VR2332, and MLV genomes.

Upregulation of heme oxygenase-1 by ginsenoside Ro attenuates lipopolysaccharide-induced inflammation in macrophage cells.

BACKGROUND: The beneficial effects of ginsenoside species have been well demonstrated in a number of studies. However, the function of ginsenoside Ro (GRo), an oleanane-type saponin, has not been sufficiently investigated. Thus, the aim of the present study was to investigate the anti-inflammatory effects of GRo in vitro using the Raw 264.7 mouse macrophage cell line treated with lipopolysaccharide (LPS), and to clarify the possible mechanism of GRo involving heme oxygenase-1 (HO-1), which itself plays a critical role in self-defense in the presence of inflammatory stress. METHODS: Raw 264.7 cells were pretreated with GRo (up to 200µM) for 1 h before treatment with 1 µg/mL LPS, and both cell viability and inflammatory markers involving HO-1 were evaluated. RESULTS: GRo significantly increased cell viability in a dose dependent manner following treatment with LPS, and decreased levels of reactive oxygen species and nitric oxide. GRo decreased inflammatory cytokines such as nitric oxide synthase and cyclooxygenase-2 induced by LPS. Moreover, GRo increased the expression of HO-1 in a dose dependent manner. Cotreatment of GRo with tin protoporphyrin IX, a selective inhibitor of HO-1, not only inhibited upregulation of HO-1 induced by GRo, but also reversed the anti-inflammatory effect of GRo in LPS treated Raw 264.7 cells. CONCLUSION: GRo induces anti-inflammatory effects following treatment with LPS via upregulation of HO-1.

Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3(-/-) and TLR4(-/-) mice, i.e. TLR3(-/-) mice were highly susceptible to JE, whereas TLR4(-/-) mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b(+)Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4(-/-) mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3(-/-) myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4(+) and CD8(+) T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b(+)Ly-6C(high) monocytes) and CD4(+)Foxp3(+) Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

Variants of the adiponectin gene and diabetic microvascular complications in patients with type 2 diabetes.

OBJECTIVE: The aim of this study was to examine the association between common polymorphisms of the adiponectin gene (ADIPOQ) and microvascular complications in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: Rs2241766 and rs1501299 of ADIPOQ were genotyped in 708 patients with T2DM. Fundus photography, nerve conducting velocity, and urine analysis were performed to check for the presence of microvascular complications including diabetic nephropathy, retinopathy and neuropathy. RESULTS: The prevalence of diabetic nephropathy tended to be different according to rs2241766 genotype (p=0.057) and the GG genotype of rs2241766 was associated with diabetic nephropathy [urine albumin/creatinine ratio (UACR) greater than 30 mg/g] after adjusting for age, sex, body mass index, duration of diabetes, HDL-cholesterol, smoking status, and blood pressure (odds ratio=1.96; 95% confidence interval=1.01-3.82, p=0.049). Also, the G allele of rs2241766 demonstrated a trend to be associated with an increase in UACR (p=0.087). Rs2241766 genotype was not associated with diabetic retinopathy (p=0.955) and neuropathy (p=0.104) or any diabetic microvascular complications (p=0.104). There was no significant association between the rs1501299 genotype of ADIPOQ and the prevalence of diabetic retinopathy and neuropathy or any diabetic microvascular complications even after adjustment. CONCLUSION: These data suggest that the GG genotype at rs2241766 is implicated in the pathogenesis of risk for diabetic nephropathy defined as UACR greater than 30 mg/day in patients with T2DM.

Gout induced by intoxication of sodium bicarbonate in Korean native broilers.

Gout is a metabolic disorder that results in hyperuricemia and the deposition of positively birefringent monosodium urate crystals in various parts of the body. Intoxication of sodium bicarbonate (SBC) for 35 days in Korean native broilers was investigated. Sixty birds, aged 2 weeks, divided into 5 groups were exposed to excess SBC: 2 g/L (group A), 7.5 g/L (group B), 20 g/L (group C), 40 g/L (group D). Toxicopathological examination of all exposed birds revealed the manifestation of visceral and articular gout in group C, while birds of group D showed acute kidney damage with manifestation of excessive visceral gout. Interestingly, few birds in group D also showed signs of rare condition of acute articular gout. Dose-dependent increments in erythrocytic count, hematocrit values, and hemoglobin levels of the exposed birds were observed. Hypernatremia, hyperuricemia, hypokalemia, and hypochloremia were common findings among exposed birds. Microscopic examination of birds that manifested visceral gout revealed significant urate deposit, tubular necrosis, and tophi formation in renal interstitium. These findings provide a pathophysiological link that SBC intoxication may support hyperuricemia, which is an independent risk factor for gout and other renal dysfunctions. Further study is required to delineate the effect of lowering uric acid on progression of gout and other renal diseases.

CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions.

CD4(+)CD25(+) regulatory T cells (T(reg)) can inhibit a variety of autoimmune and inflammatory diseases, but their involvement in regulating virus-induced immunopathology is not known. We have evaluated the role of T(reg) in viral immunopathological lesion stromal keratitis. This frequent cause of human blindness results from a T cell-mediated immunoinflammatory response to HSV in the corneal stroma. The results show that lesions were significantly more severe if mice were depleted of T(reg) before infection. The T(reg) was also shown to modulate lesion expression induced by adoptive transfer of pathogenic CD4(+) T cells in infected SCID recipients. The mechanism of T(reg) control of stromal keratitis involved suppressed antiviral immunity and impaired expression of the molecule required for T cell migration to lesion sites. Interestingly, T(reg) isolated from ocular lesions in nondepleted mice showed in vitro inhibitory effects involving IL-10, but were not very effective in established lesions. Our results decipher the in vivo role of T(reg) in a virus-induced immunopathology and imply that manipulation of regulatory cell function represents a useful approach to control viral-induced immunoinflammatory disease.