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In this study, a resistive switching (RS) memory characteristic of zinc oxide (ZnO) nanoparticles (NPs) multiple layer through a neurotransmitter and its corresponding antibody’s specific binding was demonstrated in Al/ZnO NPs/ITO device format. Particularly, a layer-by-layer (LbL) assembly was enabled through a specific immune binding between a stress hormone, cortisol, and its fittable cortisol monoclonal antibody (c-Mab). The assembly formation was accomplished using sequential LbL deposition, which could accomplish alternating self-assembly monolayers (SAMs) of ZnO NPs as the RS active layer. Here, the current–voltage (I–V) curve exhibited disparate nonvolatile RS characteristics depending on sweep polarity showing consecutively evolved hysteresis. In this study, analog-typed RS function of the ZnO NPs LbL device was demonstrated and characterized.

In this study, cortisol, which is a key stress hormone, could be detected sensitively via the colorimetric assay of a polycarbonate (PC) and glass substrate by the sandwich assay of cortisol monoclonal antibody (c-Mab) and cortisol specific binding aptamer (c-SBA). A highly sensitive change in colorimetry with a limit of detection (LOD) of cortisol of 100 fM could be attained on the optically transparent substrate using the antibody aptamer sandwich (AAS) assay by corresponding stacks of 5 nm gold nanoparticles (Au NPs). The Au NPs were conjugated by the c-SBA and the c-Mab was tethered on the PC and glass substrates. For the AAS method, a simple UV-Vis spectrophotometer was adopted to quantify the cortisol concentrations at an absorbance wavelength of 520 nm. Therefore, this study demonstrates the versatility of the AAS method to measure very low concentrations of cortisol in diagnostic applications.

Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection in type 2 diabetes patients with established cardiovascular (CV) disease independently of glycemic control. Although blood-derived coagulation factors are major contributors of CV diseases, their impact on the expression of SGLT1 and 2 in endothelial cells (ECs) has not been studied. This study investigated whether thrombin affects the expression of SGLT1 and 2 and examined their role in the induction of endothelial senescence. ECs were isolated from porcine atrial tissue (AECs) and used at first passage. Endothelial senescence was assessed by determining senescence-associated beta-galactosidase (SA-beta-gal) activity, oxidative stress using dihydroethidium, and protein level by Western blot analysis and immunofluorescence staining. Exposure of AECs to thrombin (1 or 3 U/ml) for 24 h induced an upregulation of the low protein expression level of SGLT1 and SGLT2. Thrombin induced a sustained pro-oxidant response and increased the level of SA-beta-gal activity that were both inhibited by antioxidants, an angiotensin-converting enzyme inhibitor and an AT1 receptor antagonist and also by sotagliflozin (a dual SGLT1 and SGLT2 inhibitor) and by empagliflozin (a selective SGLT2 inhibitor). In addition, thrombin upregulated the expression level of angiotensin-converting enzyme and AT1 receptors, VCAM-1 and down-regulated that of endothelial NO synthase. In conclusion, thrombin up-regulates both SGLT1 and SGLT2 expression in AECs to sustain oxidative stress leading to endothelial senescence and dysfunction. The fact that the AT1R/NADPH oxidase/SGLT1 and 2 pathway acts in a feedforward manner suggests that inhibition of SGLT1 and/or SGLT2 appears as an attractive strategy to perpetuate the protective endothelial function on the cardiovascular system. Disclosure H. Hasan: None. S. Park: None. E. Belcastro: None. C. Auger: None. H. Lee: None. V. Schini-Kerth: Research Support; Self; Boehringer Ingelheim International GmbH.

A 3-year-old male hamadryas baboon (Papio hamadryas) at the Seoul Zoo, Korea, died without any previous symptoms. Necropsy revealed severe whipworm infection in the large intestine. The animal weighed 2.6 kg and had a blood clot at the anus. Numerous whipworms were found attached to the intestinal wall, with their anterior ends embedded in the mucosa. Fecal microscopy revealed typical barrel-shaped, brown eggs of Trichuris spp., with hyaline polar plugs at each end. Histopathological examination revealed the thin anterior part of Trichuris spp. embedded in the mucosal layer and the thick posterior part at the mucosal surface or hanging freely in the intestinal lumen. This case emphasizes the importance of parasitic infection management in zoo animals.

Fibrosis is recognized as the major pathological change in adipose tissue during the development of obesity. However, the detailed mechanisms governing the interactions between the fibrotic components and their modifiers remain largely unclear. Here, we reported that matrix metalloproteinase 14 (MMP14), a key pericellular collagenase, is dramatically upregulated in obese adipose tissue. We generated a doxycycline-inducible adipose tissue-specific MMP14 overexpression model to study its regulatory function. We found that overexpression of MMP14 in the established obese adipose tissue leads to enlarged adipocytes and increased body weights in transgenic mice. Furthermore, the mice exhibited decreased energy expenditure, impaired lipid metabolism, and insulin resistance. Mechanistically, we found that MMP14 digests collagen 6α3 to produce endotrophin, a potent costimulator of fibrosis and inflammation. Unexpectedly, when overexpressing MMP14 in the early-stage obese adipose tissue, the transgenic mice showed a healthier metabolic profile, including ameliorated fibrosis and inflammation, as well as improved lipid and glucose metabolism. This unique metabolic phenotype is likely due to digestion/modification of the dense adipose tissue extracellular matrix by MMP14, thereby releasing the mechanical stress to allow for its healthy expansion. Understanding these dichotomous impacts of MMP14 provides novel insights into strategies to treat obesity-related metabolic disorders.

We describe the first case of biliary cirrhosis in Japanese macaque. Clinical signs had not been detected. The liver was nodular. Histopathologically, portal-to-portal pattern of fibrosis might have indicated chronic cholestasis. Fibrotic septa were infiltrated with inflammatory cells. Therefore, this case could be diagnosed as active incomplete biliary cirrhosis.

Background: Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence. Methods and Results: Primary ECs were isolated from porcine atrial tissue. Endothelial senescence was assessed by measuring beta-galactosidase (SA-&beta, gal) activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, protein level by Western blot, and matrix metalloproteinases (MMPs) activity using zymography. Atrial endothelial senescence was induced by thrombin at clinically relevant concentrations. Thrombin induced the up-regulation of p53, a key regulator in cellular senescence and of p21 and p16, two cyclin-dependent kinase inhibitors. Nicotinamide adenine dinucleotide phosphate NADPH oxidase, cyclooxygenases and the mitochondrial respiration complex contributed to oxidative stress and senescence. Enhanced expression levels of vascular cell adhesion molecule (VCAM)-1, tissue factor, transforming growth factor (TGF)-&beta, and MMP-2 and 9 characterized the senescence-associated secretory phenotype of atrial ECs. In addition, the pro-senescence endothelial response to thrombin was associated with an overexpression of both angiotensin converting enzyme and AT1 receptors and was inhibited by perindoprilat and losartan. Conclusions: Thrombin promotes premature ageing and senescence of atrial ECs and may pave the way to deleterious remodeling of atrial tissue by a local up-regulation of the angiotensin system and by promoting pro-inflammatory, pro-thrombotic, pro-fibrotic and pro-remodeling responses. Hence, targeting thrombin and/or angiotensin systems may efficiently prevent atrial endothelial senescence.

The number of lung cancer patients is increasing every year worldwide. For these patients' treatment, not only anticancer drugs but also immune anticancer drugs are rapidly being developed. So far, the PD-1 antibody is widely used in treating NSCLC patients, and various pharmaceutical companies are continuously developing it. However, in PD-L1 low or negative patients, new drugs are required because there are no appropriate drugs. Of course, the PD-1 antibody is sometimes used, but the response rate is low, so new therapeutic drugs are needed.We identified a new target for PD-L1 low or negative patients using PD-L1 negative or positive lung cancer tissues. The new target's code name is "Target protein A." Target protein A expressed higher in tumor than normal. As a result of analyzing caucasian lung cancer patients' tissues by immunohistochemistry, 36% of cases expressed "Target protein A". Moreover, we also confirmed that target protein A was higher in tumors than normal through the TCGA database. Based on these results, we developed an immune anticancer drug (WM-A1) targeting Target protein A and demonstrated that it induces cancer cell death by inhibiting the binding with the binding partner protein B. Meanwhile, to clarify the relationship between PD-L1 and Target protein A, we analyzed the expression between the two proteins in lung cancer cell lines and proved that they exhibit reverse correlation. Among PD-L1 low or negative lung cancer patient tissues, about 32.75% of tissues expressed Target protein A. Conversely, among the patient samples expressing Target protein A, the PD-L1 low or negative population was 63%. We checked a similar trend through the TCGA database. For the efficacy of WM-A1, we confirmed that WM-A1 induced T cell-mediated cell death in a co-culture system with human PBMC and secreted major cytokines. In addition, we revealed that the group transplanted with a cell line expressing Target protein A in the PBMC humanized model showed high immuno-anticancer efficacy. Furthermore, we demonstrated that WM-A1 increased T cell activation and related cytokine expression. However, there was no reactivity to the PD-1 antibody because it expressed Target protein A and did not express PD-L1.Therefore, we suggest that WM-A1 is a new therapeutic antibody for the PD-L1 low or negative patient population. Based on these findings, we are planning a preclinical study (GLP-Tox) in 2021. #Corresponding author: Dong-Hoon Jin*These authors (Hye jin Son and Minki Lee) contributed equally to this work. Citation Format: Hye jin Son, Minki Lee, Seongrak Kim, Jai-Hee Moon, Hana Kim, Wonhwa Shin, Joseph Kim, Mi So Lee, Daeun Kim, Seunggeon Bae, Joonyee Jeong, Seung-Woo Hong, Chun-Ho Park, Hyun Ho Lee, Dong-Hoon Jin. New therapeutic antibody ("WM-A1") for treatment of low or no PD-L1 NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1216.

Fine dust (FD) is a form of air pollution and is responsible for a wide range of diseases. Specially, FD is associated with several cardiovascular diseases (CVDs); long-term exposure to FD was shown to decrease endothelial function, but the underlying mechanism remains unclear. We investigated whether exposure to FD causes premature senescence-associated endothelial dysfunction in endothelial cells (ECs) isolated from porcine coronary arteries. The cells were treated with different concentrations of FD and senescence associated-beta galactosidase (SA-β-gal) activity, cell cycle progression, expression of endothelial nitric oxide synthase (eNOS), oxidative stress level, and vascular function were evaluated. We found that FD increased SA-β-gal activity, caused cell cycle arrest, and increased oxidative stress, suggesting the premature induction of senescence; on the other hand, eNOS expression was downregulated and platelet aggregation was enhanced. FD exposure impaired vasorelaxation in response to bradykinin and activated the local angiotensin system (LAS), which was inhibited by treatment with the antioxidant N-acetyl cysteine (NAC) and angiotensin II receptor type 1 (AT1) antagonist losartan (LOS). NAC and LOS also suppressed FD-induced SA-β-gal activity, increased EC proliferation and eNOS expression, and improved endothelial function. These results demonstrate that FD induces premature senescence of ECs and is associated with increased oxidative stress and activation of LAS. This study can serve as a pharmacological target for prevention and/or treatment of air pollution-associated CVD.

Background Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that can infect warm-blooded animals including humans. New World monkeys, such as squirrel monkeys, are more susceptible to T. gondii than Old World monkeys, often developing fatal disease. Methods In this study, seven of thirteen dead squirrel monkeys at Seoul Grand Park were tested to find the cause of sudden death. Results The main histopathological findings included interstitial pneumonia, necrotizing hepatitis, and splenitis. Periodic acid-Schiff staining of liver, spleen, and lung revealed cyst structures consistent with bradyzoites. Amplification of the B1 gene was detected in the liver or spleen of all monkeys. Additionally, a restriction fragment length polymorphism assay and phylogenetic analysis of the GRA6 amplicon revealed a consistent clustering with the type II strain of T. gondii. Conclusions This study is the first report of T. gondii infection of squirrel monkeys in Korea, and the first report of type II T. gondii based on GRA6 analysis in Korea.

Objective After injury to the central nervous system (CNS), glial scar tissue is formed in the process of wound healing. This can be is a clinical problem because it interferes with axonal regeneration and functional recovery. It is known that intracellular proteins, including the glial fibrillary acidic protein (GFAP), nestin, and vimentin increase in the astrocytes after an injury to the CNS. By studying the time course and co-expression pattern of these intracellular proteins, this study will attempt to prove that these proteins are involved in the processes of glial scar formation. Methods Twenty-five male Sprague-Dawley rats were used in this study. Bregma of the cerebral cortex, an area was incised with a sharp blade, and perfusion was performed. The expressions of the intracellular proteins were assayed, while the co-localization of the intermediate filament (GFAP, nestin, and vimentin) and A2B5 were examined. Results At 12 hours, the GFAP was expressed in the white matter underlying the lesion, and in the cerebral cortex. Nestin was expressed in the astrocytes in the perilesional area after 3 days, while A2B5 was observed in the edge of the wound at 12 hours post-injury, with its expression reaching a peak at 7 days. Vimentin was detected in the white matter at 12 hours, and in the cortex, reaching a peak at 7 days. Conclusion In the processes of glial scar formation, nestin, vimentin, and A2B5 were revealed in the astrocytes, and these factors may be involved in the division, proliferation, and transportation of the astrocytes.

We investigated the production of influenza A/Korea/01-2-9/2009 (H1N1) virus-like particles (VLPs) containing four structural proteins, matrix protein 1 (M1), matrix protein 2 (M2), neuraminidase (NA) and hemagglutinin (HA), using stably transfected Trichoplusia ni BT1 TN-5B1-4 (TN-5B1-4) cells. Recombinant M1, M2, NA and HA were expressed as bands with molecular weights of 28, 17, 60, and 70 kDa, respectively, in stably transfected TN-5B1-4 (TN-5B1-4/M1-M2-NA-HA) cells. VLPs were purified from the culture medium of the TN-5B1-4/M1-M2-NA-HA cells by pelleting on a sucrose cushion, followed by ultracentrifugation in a sucrose density gradient. The four structural proteins were released together from the TN-5B1-4/M1-M2-NA-HA cells, and were co-purified from the same fractions of the sucrose density gradient, indicating that recombinant M1, M2, NA, and HA self-assembled into VLPs in the TN-5B1-4/M1-M2-NA-HA cells. Recombinant baculoviruses (rBac/NA and rBac/HA) expressing recombinant NA and HA were generated and used to co-infect stably transfected TN-5B1-4 (TN-5B1-4/M1-M2) cells expressing recombinant M1 and M2, resulting in the production of high-molecular-weight VLPs and the release of self-assembled VLPs with diameters of 80 ∼ 120 nm. The production of VLPs was greatly enhanced in the TN-5B1-4/M1-M2 cells co-infected with rBac/NA and rBac/HA, compared with the TN-5B1-4/M1-M2-NA-HA cells. Our results suggest that a stably transfected-insect cell expression system might be useful for producing VLPs for the development of recombinant vaccines against influenza.

A 19-year-old captive male waterbuck (Kobus ellipsiprymnus) exhibited traumatic pericarditis at necropsy. The animal weighed 182 kg at necropsy and revealed no remarkable findings in external observation. Severe pericardial adhesions with fibrosis, hepato-diaphragmatic adhesions, straw-colored ascites and hepatosplenomegaly were observed upon examining the internal organs. Perforations made by a 12-cm-long sharp-ended bamboo twig were detected in the reticulum, diaphragm, pericardium, lung and liver. Trueperella pyogenes was identified in pericardial fluid. To our knowledge, this is the first documented case of traumatic reticulopericarditis caused by a sharp-ended bamboo twig in a captive waterbuck.

The exact mechanism of cadmium (Cd) immobilization by oyster shell (OS) has not been reported. The effect of OS on Cd immobilization and the exact mechanism should be known before applying remediation technology using OS to Cd contaminated soils. Therefore, the objective of this study was to elucidate the mechanism of Cd immobilization by OS. Three grams of OS (

Bacillus cereus is food poisoning bacteria frequently occured in starch food. Most of the delivery foods for infant is classified as ready-to-cook food. But unlike food for infant and young children, there are no stan- dards and specifications of Bacillus cereus in ready-to-cook food. The purpose of this study is to examine the presence of Bacillus cereus, aerobic bacteria and coliforms in the food for infant and young children sold through internet. B. cereus was detected in 9 samples (8.3%), total aerobic bacteria was detected over 10 6 CFU/g in 4 samples and coliforms were not detected in any samples. This will provide basic data for standards and specifications of Bacillus cereus in ready-to-cook food.

Three recombinant polypeptides, VP1-His, VP1-3N-His, and 3D2-His, were produced by Escherichia coli expression system. Recombinant VP1-His, VP1-3N-His, and 3D2-His were expressed as bands with molecular weights of 32, 38, and 30 kDa, respectively. These were purified by affinity chromatography using Ni-NTA Fast-flow resin and/or ion-exchange chromatography using DEAE-Sepharose Fast-flow resin. Intraperitoneal immunizations of recombinant polypeptides successfully elicited the productions of VP1-His, VP1-3N-His, and 3D2-His specific IgG antibodies (IgG subclass distribution of IgG1>IgG2a>IgG2b>IgG3) in sera and induced the secretions of cytokines IFN-γ and IL-6 in spleen cells. Sera from recombinant VP1-His-, VP1-3N-His-, and 3D2-His-immunized mice neutralized the propagation of HAV. The highest neutralizing activity was shown in sera from recombinant VP1-3N-His-immunized mice. These results suggest that recombinant VP1-3N-His can be a useful source for developing hepatitis A virus (HAV) subunit vaccine candidates.

Transgenic tomato plants expressing the gene of a chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus (HAV) VP1 and an Fc antibody fragment have been obtained. Recombinant VP1-Fc protein with a molecular mass of approximately 68 kDa was purified from transgenic tomato plants using Protein A Sepharose affinity chromatography. The recombinant protein elicited production of specific IgG antibodies in the serum after intraperitoneal immunization of BALB/c mice. The antibodies produced by mice against transgenic plant-derived recombinant VP1-Fc most likely recognize epitopes in the HAV viral antigen. Following vaccination with recombinant VP1-Fc protein, expression of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Our findings indicate that transgenic tomato plants can provide a useful system for the production of HAV antigens.

Bloomless cucumber fruits are commercially produced by grafting onto the pumpkin stocks (Cucurbita moschata ) to restricted silicon (SiO₂) absorption. Inhibition of silicon absorption in bloomless stocks is conferred by a mutant allele of the CmLsi1 homologous to Lsi1 in rice. In this study, we characterized the Lsi1 homologs in pumpkin (C. moschata ) and its coldtolerant wild relative C. ficifolia (‘Heukjong’) in order to develop a DNA marker for selecting a bloomless trait and to establish the molecular basis for breeding bloomless stock cultivars of C. ficifolia . A Cleaved amplified polymorphic sequence (CAPS) marker (CM1-CAPS) was designed based on a non-sysnonymous single nucleotide polymorphism (SNP, C＞T) of the CmLsi1 mutant-type allele, and its applicability for Marker-assisted selection (MAS) was confirmed by evaluating three bloom and five bloomless pumpkin stock cultivars. Quantitative RT-PCR of the CmLsi1 for these stock cultivers implied that expression level of the CmLsi1 gene does not appear to be associated with the bloom/bloomless trait and may differ depending on plant species and tissues. A full length cDNA of the Lsi1 homolog [named CfLsi1 (B⁺)] of ‘Heukjong’ (C. ficifolia ), was cloned and sequence comparison between CmLsi1 (B⁺) and CfLsi1 (B⁺) revealed that there exists total 24 SNPs, of which three were non-synonymous. Phylogenetic analysis of CfLsi1 (B⁺) and Lsi1 homologs further revealed that CfLsi1 (B⁺) is closesly related to Nodulin 26-like intrinsic proteins (NIPs) and most similar to CpNIP1 of C. pepo than C. moschata.

Vivax malaria is a significant military and civilian health threat in northern Republic of Korea (ROK). Mosquito collections were performed at two ROK army installations, Paju near the demilitarized zone (DMZ) using black light traps in 2011. The DMZ, a 4 km wide border, is the northernmost point of the ROK and separates the ROK from the Democratic People's Republic of Korea (DPRK). Anopheles spp. were identified by polymerase chain reaction and screened for Plasmodium vivax sporozoites. Of 4,354 female Anopheles mosquitoes identified, Anopheles kleini (61.8%) was the most frequently collected, followed by Anopheles pullus (16.0%), Anopheles belenrae (9.0%), Anopheles sinensis (7.4%), Anopheles sineroides (4.2%), and Anopheles lesteri (1.6%). Anopheles kleini, An. pullus, and An. sineroides showed the highest population densities in June, whereas population densities were highest for An. belenrae, An. lesteri, and An. sinensis in August. The maximum likelihood estimation (estimated number of positive mosquitoes/1,000) for P. vivax was highest for An. lesteri (28.9), followed by An. sineroides (23.3), An. belenrae (15.8), An. sinensis (9.6), An. pullus (5.8) and An. kleini (4.2). The seasonal maximum likelihood estimation (MLE) values were variable among Anopheles species. Anopheles belenrae, An. Pullus, and An. sineroides showed the highest seasonal MLE's in July, whereas An. lesteri and An. sinensis exhibited the highest seasonal MLEs in September and An. kleini during August. This is the first report implicating An. sineroides as a vector of P. vivax in the ROK, and extends our knowledge of the distribution and potential role in malaria transmission.

Hypercholesterolemia is a well-known etiological factor for cardiovascular disease and a common symptom of most types of metabolic disorders.Camellia japonicais a traditional garden plant, and its flower and seed have been used as a base oil of traditional cosmetics in East Asia. The present study was carried out to evaluate the effect ofC. japonicafruit extracts (CJF) in a high fat diet- (HFD-) induced hypercholesterolemic rat model. CJF was administered orally at three different doses: 100, 400, and 800 mg·kg−1·day−1(CJF 100, 400, and 800, resp.). Our results showed that CJF possessed strong cholesterol-lowering potency as indicated by the decrease in serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL), accompanied by an increase in serum high-density lipoprotein (HDL). Furthermore, CJF reduced serum lipid peroxidation by suppressing the formation of thiobarbituric acid reactive substance. In addition, oil red O (ORO) staining of rat arteries showed decreased lipid-positive staining in the CJF-treated groups compared to the control HFD group. Taken together, these results suggest that CJF could be a potent herbal therapeutic option and source of a functional food for the prevention and treatment of atherosclerosis and other diseases associated with hypercholesterolemia.

BACKGROUND Homelessness is associated with an increased risk of exposure to Mycobacterium tuberculosis. Several factors, including alcoholism, malnutrition, lack of stable housing, combine to make tuberculosis more prevalent in the homeless. The aims of this study were to determine the factors associated with increasing success rate of tuberculosis treatment in the homeless. METHODS A cross-sectional analysis of the clinical features in 142 pulmonary tuberculosis-positive homeless patients admitted to the Busan Medical Center from January 2001 to December 2010 was carried out. These results were compared with a successful treatment group and incomplete treatment group. We also evaluated the risk factors of treatment non-completion. Statistical analysis for the comparisons was performed using a χ(2) test, independent samples t-test, and multiple logistic regression. RESULTS Comparison of clinical characteristics showed significant differences between the two groups in the type of residence (P < 0.001), diseases with risk factors (P = 0.003), and history of tuberculosis treatment (P = 0.009). Multiple regression analysis revealed the residence (odds ratio [OR], 4.77; 95% confidence interval [CI], 2.05 to 11.10; P < 0.001) and comorbidity with risk factor (OR, 2.72; 95% CI, 1.13 to 6.53; P = 0.025) to be independently associated with treatment success. CONCLUSION To improve the success rate of tuberculosis treatment in the homeless person, anti tuberculosis medication should be taken until the end of treatment and a management system for the homeless person is required. Further social and medical concerns for stable housing and management of comorbidity may lead to an improvement in the successful tuberculosis treatment of homeless person.

Dimethyl sulfoxide (DMSO) increased the intracellular calcium ion concentration in stably transfected Drosophila melanogaster S2 cells expressing recombinant cyclooxygenase 1 (COX-1). DMSO did not increase the Drosophila NOS (dNOS) transcript level in calcium chelatortreated cells. Expression of recombinant COX-1 due to DMSO was diminished in cells treated with calcium chelators or channel blockers. Our results indicate that an increased intracellular calcium ion concentration due to DMSO is associated with up-regulation of the dNOS gene, leading to enhanced expression of COX-1.

Recombinant human cyclooxygenase 1 (COX-1) was expressed from stably-transfected Drosophila melanogaster S2 (S2) cells. DMSO improved the expression of recombinant COX-1 by 180 %. DMSO increased the expression of nitric oxide synthase (NOS) at both the RNA and protein levels; NOS expression was closely correlated with the synthesis of recombinant COX-1 mRNA in stably-transfected S2 cells. DMSO also induced the gene encoding Kr-h1 which binds to the CACCC element of the metallothionein promoter to enhance the expression of recombinant COX-1. Therefore, DMSO improves the expression of recombinant COX-1 via NOS and/or the transcription factor Kr-h1.

We describe expression and characterization of recombinant human colorectal cancer antigen GA733-2 fused to Fc fragment of antibody (GA733-2-Fc) using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves. Recombinant GA733-2-Fc/KDEL with a molecular mass of ~68 kDa was transiently expressed. The level of expression of GA733-2-Fc with ER retention signal KDEL (GA733-2-Fc/KDEL) in the expression vector system was 0.96% of total soluble proteins. Recombinant GA733-2-Fc/KDEL was purified using an affinity chromatography. Mice immunized with recombinant GA733-2-Fc/KDEL mounted a strong GA733-2-Fc/KDEL-specific serum antibody response. Vaccination of plant-derived recombinant GA733-2-Fc/KDEL regressed tumor volumes in BALB/c mice. The population of activated-T and NK-T cells increased notably in lymph node, spleen, and tumor-infiltrating lymphocytes derived from the tumor-regressed mice. Taken together, recombinant GA733-2-Fc/KDEL expressed in plants can be used as an effective experimental immunogen for research in cancer vaccine development.

Lung cancer is second most common cancer in the world. Non Small Cell Lung Cancer (NSCLC) accounts approximately 80-85% of all lung cancer diagnosis. Traditional therapies of this disease were surgical resection, chemotherapy, and radiotherapy, alone or in combination. In addition, targeted therapeutic approach was based on the concept of discovering genetic alterations and the signaling pathways in cancer. Recently, to overcome the critical points for standard therapies, many groups were studied immunotherapeutic approaches, such as programed cell-death protein 1 (PD-1) antibody. However, rational use of these agents has been limited by the lack of a definitive predictive biomarker. Therefore, we identified new target, cancer immunotherapy related gene, CMG by shRNA libraries screening analysis on chemo-agents & target therapy resistant non-small cell lung cancer cells. First of all, we investigated CMG expression by immunohistochemistry in various tissue microarray (TMA). These results show that CMG highly expressed in Lung cancer, Liver cancer, and gastric cancer. We investigated target potentials on lung cancer, liver cancer, and gastric cancer cell lines using in vitro and in vivo assay system. Knockdown of CMG by CMG shRNA was induced cell death in various cancer cell lines. In addition, suppression of CMG was induced tumor size regression in CMG shRNA stable cell lines-derived xenograft model. Based on these results, we synthesized a novel series of CMG therapeutic antibody. CMG therapeutic antibody is a lead antibody for treating Lung cancer patients who express CMG gene. These antibodies have anti-cancer effects and immunotherapeutic effects in lung cancer (NSCLC), liver cancer, and gastric cancer. In addition, the in vivo efficacy of CMG antibody was assessed in mouse lung cancer derived syngeneic mouse model. The CMG antibody was tri-daily i.p. injected and the tumor volume was measured and compared between groups. Dramatic tumor regression was observed in CMG antibody treated group. These results were shown that these antibodies have immunotherapeutic potentials. In conclusion, CMG is a promising target for Lung cancer patients (chemo-agents resistant or PD-1 resistant Lung cancer patienrts). Our antibodies can be promising therapeutic agents for lung cancer, Liver cancer, and gastric cancer. Citation Format: Jai-hee Moon, Dae Hee Lee, Jae-Sik Shin, Joseph Kim, Yoon Sun Park, Seung-Woo Hong, So Hee Lee, Mi Jin Kim, Joonyee Jung, Chun-Ho Park, Sun-Chul Hur, Hyojin Kim, Hyebin Park, Sang Soo Park, Jun Ki Hong, Ji Hee Gong, Jieun Kim, Hyun Ho Lee, Il-Whea Ku, Dong-Hoon Jin. Development of new mechanism based therapeutic antibodies in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2909.

Human colorectal cancer antigen GA733-2 fused to the immunoglobin Fc fragment (GA733-2-Fc) was expressed in stably transformed Drosophila melanogaster S2 cells, and the immunogenicity of recombinant GA733-2-Fc was investigated in mice. Recombinant GA733-2-Fc was secreted into a culture medium with a molecular mass of approximately 65 kDa. Recombinant GA733-2-Fc was purified to homogeneity using affinity fractionation with Protein A sepharose 4 Fast Flow. Recombinant GA733-2-Fc proteins elicited production of specific antibodies against recombinant GA733-2 by immunization through an intraperitoneal route. Recombinant GA733-2-Fc-induced antibodies showed a binding activity to human colorectal carcinoma HCT-116 cells. Secretory recombinant GA733-2-Fc from Drosophila S2 cell systems can be used as an effective experimental antigen for research in cancer vaccine development.

A lab-on-a-chip for the rapid identification of microbial species has been developed for a water monitoring system. We employed highly parallel DNA microarrays for the direct profiling of microbial populations in a sample. For the integration and minimization of the DNA microarray protocols for bacterial identification, rRNA was selected as a target nucleotide for probe:target hybridization. In order to hybridize target rRNA onto the probe oligonucleotide, intact rRNA extracted fromE. coli rRNA was fragmented via chemical techniques in the lab-on-a-chip platform. The size of fragmented rRNA was less than 400 base pairs, which was confirmed by polyacrylamide gel electrophoresis. The fragmented rRNA was also labeled using fluorescent chemicals. The lab-on-a-chip for fragmentation and labeling includes a PDMS chaotic mixer for efficient mixing, operated by flow pressure. In addition, the fragmented rRNA was hybridized successfully on a DNA microarray with sample recirculation on a microfluidic platform. Our fragmentation and labeling technique will have far-reaching applications, which require rapid but complicated chemical genetic material processing on a lab-on-a-chip platform.

Camellia japonicais a popular garden plant in Asia and widely used as cosmetic sources and traditional medicine. However, the possibility thatC. japonicaaffects cardiovascular system remains unclear. The aim of the present study was to evaluate vascular effects of an extract ofC. japonica. Vascular reactivity was assessed in organ baths using porcine coronary arteries and inhibition of proliferation and migration were assessed using human vascular smooth muscle cells (VSMCs). All four different parts, leaf, stem, flower, and fruits, caused concentration-dependent relaxations andC. japonicafruit (CJF) extract showed the strongest vasorelaxation and its effect was endothelium dependent. Relaxations to CJF were markedly reduced by inhibitor of endothelial nitric oxide synthase (eNOS) and inhibitor of PI3-kinase, but not affected by inhibitor of cyclooxygenase and endothelium-derived hyperpolarizing factor-mediated response. CJF induced activated a time- and concentration-dependent phosphorylation of eNOS in endothelial cells. Altogether, these studies have demonstrated that CJF is a potent endothelium-dependent vasodilator and this effect was involved in, at least in part, PI3K-eNOS-NO pathway. Moreover, CJF attenuated TNF-αinduced proliferation and PDGF-BB induced migration of VSMCs. The present findings indicate that CJF could be a valuable candidate of herbal medicine for cardiovascular diseases associated with endothelial dysfunction and atherosclerosis.

We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

Microbial desulfurization of low rank coal by Rhodococcus rhodochrous IGTS8 was investigated using three different pretreated coal samples. Solubilized coal was desulfurized more efficiently than hard coal and more sulfur was extracted from biologically solubilized coal than from chemically solubilized coal. Microbial desulfurization combined with biological solubilization removed 75% of the total sufur while the microbial desulfurization combined with chemical solubilization removed 63%.

Introduction The beneficial cardiovascular effect of polyphenol-rich food and beverages has been attributed, at least in part, to an improved vascular function through an increased endothelial formation of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), two major vasoprotective mechanisms. Black currant is a rich source of anthocyanins, and, in particular, of glucoside- and rutinoside-conjugated delphinidin and cyanidin. This study evaluated the role of sodium-glucose cotransporters (SGLT) 1 and 2 in the blackcurrant extract (BCE)-induced NO-mediated endothelium-dependent relaxation and activation of endothelial NO synthase (eNOS). Methods The reactivity of porcine coronary artery rings was assessed in organ chambers, and the expression and phosphorylation level of proteins in cultured porcine coronary artery endothelial cells by Western blot analysis. Results BCE (310.0 ± 0.1 mg gallic acid equivalent/g) was prepared from a blackcurrant juice containing 2.7 g gallic acid equivalent/L of polyphenols using a Sephadex LH-20 column. BCE caused potent concentration-dependent relaxations of coronary artery rings with endothelium, which were significantly reduced by the dual SGLT1 and SGLT2 inhibitor LX4211, and also to some extent by the SGLT2 inhibitor canagliflozin but not dapagliflozin. In contrast, LX4211 did not affect relaxations to bradykinin, sodium nitroprusside and epigallocatechin gallate. BCE induced the phosphorylation of eNOS at the activator site Ser1177 in cultured endothelial cells, which was significantly prevented by LX4211, dapagliflozin and canagliflozin. Conclusion These observations indicate that BCE is a potent activator of eNOS and inducer of NO-mediated vasorelaxation possibly subsequent to the entry of conjugated anthocyanins via SGLT1/2 into endothelial cells.

Introduction Adansonia digitata leaves are used for the traditional treatment of hypertension in Senegal. This study evaluated the relaxant effect of a hydroethanolic extract of leaves of Adansonia digitata (ADF) in artery rings, and investigated the underlying mechanism. Methods Vascular reactivity was assessed in organ chambers. Rings were either untreated or incubated with a pharmacological tool for 30 min before contraction of coronary artery rings with either U46619 or phenylephrine, and the subsequent addition of ADF. In some experiments, the endothelium was removed mechanically. The phosphorylation level of target proteins was assessed by Western blot analysis. Results ADF induced concentration-dependent relaxations in coronary artery rings with endothelium, which reached 100% at a concentration as low as 10 μg/ml. The relaxation to ADF was significantly reduced by NLA (NO synthase inhibitor) and by the combination of Tram-34 and apamin (inhibitors of intermediate and small conductance calcium-dependent K+ channels, respectively), not affected by indomethacin (cyclooxygenase inhibitor), and abolished by wortmannin (PI3-kinase inhibitor) and MnTMPyP (membrane permeant superoxide dismutase mimetic). ADF caused relaxations in the rat superior mesenteric artery, carotid artery and aortic rings but not in the secondary mesenteric artery and femoral artery rings. ADF induced a sustained phosphorylation of Akt and eNOS at Ser1177 in cultured porcine coronary artery endothelial cells. Conclusion The Adansonia digitata leave extract induced potent endothelium-dependent relaxations, involving predominantly NO and endothelium-dependent hyperpolarization. Such vasorelaxant effects may explain the antihypertensive use of this plant in traditional African medicine.

Streptococcus mutans and Streptococcus sobrinus are the major causative agents of human dental caries. Therefore, the removal or inhibition of these streptococcal biofilms is essential for dental caries prevention. In the present study, we evaluated the effects of ribose treatment alone or in combination with xylitol on streptococcal biofilm formation for both species. Furthermore, we examined the expression of genes responsible for dextran-dependent aggregation (DDAG). In addition, we investigated whether ribose affects the biofilm formation of xylitol-insensitive streptococci, which results from long-term exposure to xylitol. The viability of streptococci biofilms formed in a 24-well polystyrene plate was quantified by fluorescent staining with the LIVE/DEAD bacterial viability and counting kit, which was followed by fluorescence activated cell sorting analysis. The effects of ribose and/or xylitol on the mRNA expression of DDAG-responsible genes, gbpC and dblB, was evaluated by RT-qPCR. Our data showed that ribose and other pentose molecules significantly inhibited streptococcal biofilm formation and the expression of DDAG-responsible genes. In addition, co-treatment with ribose and xylitol decreased streptococcal biofilm formation to a further extent than ribose or xylitol treatment alone in both streptococcal species. Furthermore, ribose attenuated the increase of xylitol-insensitive streptococcal biofilm, which results in the reduced difference of biofilm formation between S. mutans that are sensitive and insensitive to xylitol. These data suggest that pentose may be used as an additive for teeth-protective materials or in sweets. Furthermore, ribose co-treatment with xylitol might help to increase the anti-cariogenic efficacy of xylitol.

We describe the expression and immunogenicity of a recombinant chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus VP1 and an Fc antibody fragment using a replicating vector based on Beet curly top virus (BCTV) in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Recombinant HAV VP1-Fc was expressed with a molecular mass of approximately 68 kDa. Recombinant HAV VP1-Fc, purified using Protein A Sepharose affinity chromatography, elicited production of specific IgG antibodies in the serum after intraperitoneal immunization. Following vaccination with recombinant HAV VP1-Fc protein, expressions of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Recombinant VP1-Fc from infiltrated tobacco plants can be used as an effective experimental immunogen for research into vaccine development.

This paper reports a multi-walled carbon nanotube (MWNT) cluster-based process for removing bacteria by uniquely combining the two excellent properties of carbon nanotubes (CNTs): magnetic susceptibility and bacterial affinity. The reaction system consisted of large clusters of MWNTs for bacterial capture and an external magnet for bio-separation. The designed system was tested using four different bacteria strains, including both gram-positive and gram-negative bacteria. The results demonstrated CNTs' excellent potential as highly effective bacterial adsorbents for the spontaneous adsorption of any types of bacteria to the clusters and as highly effective paramagnetic complexes for the rapid and highly effective magnetic separations. The CNT based system would be beneficial to many biomedical as well as environmental applications that require bacterial removal, including antimicrobial treatment, graft and implant purification, water purification, food processing, and biosensing of pathogens.

We describe the secretory expression and immunogenicity of the recombinant HAV (hepatitis A virus) structural polypeptide VP1 from stably transformed Drosophila melanogaster S2 (Schneider 2) cells. Southern-blot analysis indicated that transformed S2 cells contained multiple copies of the HAV VP1 gene in the genome. Recombinant VP1 was secreted into a culture medium with a molecular mass of 42-49 kDa. A maximum production level of 6.24 mg of recombinant VP1/litre was obtained in a T-flask culture of Drosophila S2 cells 5 days after induction with 0.5 mM CuSO4. The recombinant HAV VP1 protein elicited the production of specific IgA in the small intestine by oral immunization and production of specific IgG in the serum by intraperitoneal immunization. Our findings show that secretory recombinant VP1 from transformed Drosophila S2 cells can be used as an effective experimental immunogen for research in vaccine development.

Four new chalcones 1 - 4, named abyssinones A - D, were isolated from the stem bark of the plant Erythrina abyssinica and their structures were elucidated on the basis of spectroscopic analyses. The compounds 1, 3, and 4 were found to exhibit moderate cytotoxic activity against the human colorectal cancer cell line (Caco2) with IC (50) values of 13.3, 15.1, and 11.1 microM, respectively.

IgE-mediated allergic reactions caused by mosquito bites are a common problem all over the world. This study was undertaken to determine IgE levels in subjects, to elucidate human IgE and mouse IgG1 binding patterns and to investigate the cross-reactivity of salivary gland antigens with three mosquitoes. Mosquito larvae of Aedes togoi, Culex tritaeniorhynchus and Culex pipiens pallens were collected and maintained in the laboratory. Salivary gland extracts (SGE) and whole-body extracts (WBE) were prepared from female mosquitoes of each species. The 9 sera out of 12 with positive skin reactions to SGE of A. togoi by skin prick test showed significantly higher anti-mosquito SGE IgE levels than in those without skin reactions. Protein band patterns of the SGE and WBE of the three species were different from one another. There were specific mouse IgG1 reactions to the bands of 30.5, 33, 37 and 57.5 kD in the SGE of A. togoi. The ELISA inhibition studies disclosed almost no crossreactivities between A. togoi, C. tritaeniorhynchus and C. pipiens pallens. Immunoblot analysis disclosed that allergenic proteins in the SGE of mosquitoes and their patterns were remarkably similar between human and mouse sera to the SGE of A. togoi. Species shared allergens may not exist among the three mosquito species prevalent in Korea.

The effect of DMSO and sodium butyrate on the production of recombinant hepatitis A virus (HAV) capsid protein VP1 was evaluated and optimized in the culture of stably transfected Drosophila melanogaster S2 cells using culture plates and spinner flasks. The effect of DMSO and sodium butyrate was also evaluated to improve the recombinant VP1 production in stably transfected Drosophila S2 cells. A production level of 0.88 ㎎ of recombinant VP1/liter was obtained in the culture-plate culture of stably transfected S2 cells at 6 days after induction with 0.5 mM CuSO4. The supplements of 2% DMSO and 10 mM sodium butyrate at 4 days post-inoculation increased recombinant VP1 accumulation by 141 and 104%, respectively, resulting in 2.17 and 1.7 ㎎/liter of recombinant VP1 production. In spinner flasks, recombinant VP1 production reached maximum level at 9 days after induction with 0.5 mM CuSO4, with approximately 4.96 ㎎/liter of recombinant VP1 production level. When 2% DMSO or 10 mM sodium butyrate was added at 5 days post-inoculation, the recombinant VP1 production was increased to 8.35 and 5.85 ㎎/liter, respectively. However, the synergistic effects of DMSO and sodium butyrate were not observed. These results indicate that DMSO and/or sodium butyrate can be successfully used to improve the recombinant HAV VP1 production in culture plates and spinner flasks.

A nuclear polyhedrosis virus ofHyphantriacunea replicated successfully in theTrichoplusiani cell line. Restriction endonuclease analysis of the viral DNA obtained from infected cell culture showed the same general homogeneity as that from virus isolated from diseased host larvae. Electron microscopic observations showed that the occluded virus from cell culture consists of rod-like nucleocapsids (31×320 nm) enveloped in aggregates and embedded in polyhedral inclusion bodies from 0.6 to 2.5 μm in diameter.