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The protective effect of Leucosporidium-derived ice-binding protein (LeIBP) on bovine oocytes and embryos during vitrification
No full textIce-binding proteins (IBPs) facilitate organism survival under extreme conditions by inhibiting thermal hysteresis and ice recrystallization. IBPs have been widely used as cryoprotectants to cryopreserve mammalian gametes and embryos. In the present study, we evaluated the protective effects of an Arctic yeast, Leucosporidium sp. AY30 derived ice-binding protein (LeIBP), on the vitrification of bovine metaphase II (MII) oocytes and embryos. When oocytes and embryos were frozen using the two-step vitrification method, the survival rate was significantly increased in the presence of LeIBP. The LeIBP supplementation decreased the levels of intracellular reactive oxygen species (ROS) and enhanced mitochondrial functions in the vitrified？warmed oocytes. Furthermore, LeIBP improved the developmental potential and suppressed apoptosis of the embryos derived from vitrified？warmed oocytes. Collectively, these data indicate that LeIBP can be used as a promising cryoprotectant to prevent cryoinjury during vitrification in bovine oocytes. ⓒ 2020 The Authors
Microfluidic immunoassay for point-of-care testing using simple fluid vent control
No full textMicrofluidic immunoassays have gained immense importance as a point-of-care testing (POCT) platform owing to their low reagent volume requirements, low cost, portability, and rapid reaction. However, they are characterized by inconsistency and lack of reproducibility. In this study, we developed a microfluidic immunoassay platform with a simple signal enhancement technique using fluid vent control. The components of this fluorescence-based immunoassay were successfully pre-loaded in the microfluidic device. The analytes reacted with the detection antibody conjugated on fluorescence beads, and bound to capture the antibody immobilized zone during channel flow. The fluid vent was closed using a small PC-connected equipment when the fluid reached the target displacement. The pausing of the fluid provided sufficient time for the immune reaction, and this addition of the fluid pausing significantly enhanced the immune reaction and increased the limit of detection (LOD) of the immunoassay. The LOD of the developed assay was the same as that of the sandwich fluorescence immunoassay in terms of quantity. Furthermore, the LOD was significantly higher than that of the commercial rapid kits. Therefore, our simple technique could be employed for cost-effective and accurate immunoassays in the field of POCT.
Surface-functionalized 3D porous rGO-polysaccharide sphere composites for rapid selective protein adsorption from crude biological liquid
No full textA novel protein adsorbent was developed by the surface modification of porous rGO-polysaccharide sphere composites with Coomassie Brilliant Blue G250 (CBB-G250). The advanced protein adsorbent combined selectively with proteins from crude bacteria lysate within 10 min, while deoxyribonucleic acid (DNA) was not adsorbed. Thus, protein and DNA in complicated biological samples were separated very quickly and easily during mild mixing treatment with the surface-functionalized rGO-polysaccharide sphere composites. The rGO in the porous sphere composites plays a key role as a linker in the adsorption of CBB-G250 on the composites and makes the CBB-G250 dye bind protein selectively from crude samples. It was confirmed that selective protein adsorption was only possible when the CBB-G250 dye had a chance of interaction with rGO. In addition, the CBB-G250-functionalized rGO-polysaccharide sphere composites could capture whole pathogens because of binding to the surface proteins of the pathogens. Therefore, the surface-functionalized rGO-polysaccharide sphere composites are good candidates for application in simple and efficient methods for the separation and purification of DNA in crude pathogen lysate and for the removal of whole pathogens by selective binding with proteins in the area of rapid pathogen detection and diagnosis.
Fanconi anemia pathway activation by FOXM1 is critical to bladder cancer recurrence and anticancer drug resistance
No full textAlthough the 5-year survival rate of patients diagnosed with nonmuscle invasive bladder cancer (NMIBC) has reached 85%, more than 50% of patients suffer from frequent recurrences. To identify molecular targets associated with recurrence of NMIBC, we analyzed gene expression data and found that FOXM1 and FANCD2 were involved in recurrence. Therefore, we investigated how these genes were involved in the mechanism of recurrence and confirmed their usefulness as biomarkers. Investigation have shown that FOXM1 directly regulated the transcription of FANCD2, which is the key gene of the Fanconi anemia (FA) pathway. Depletion of FOXM1 resulted in DNA repair defects in the FA pathway and in decreased resistance to chemotherapy. Thus, the FANCD2-associated FA pathway activated by FOXM1 is an important mechanism involved in chemotherapy-related recurrence. In conclusion, FOXM1 and FANCD2 can be used as prognostic factors that are associated with high risk of recurrence and with anticancer drug resistance properties in NMIBC patients.
Synaptic loss and amyloid beta alterations in the rodent hippocampus induced by streptozotocin injection into the cisterna magna
No full textTo date, researchers have developed various animal models of Alzheimer's disease (AD) to investigate its mechanisms and to identify potential therapeutic treatments. A widely recognized model that mimics the pathology of human sporadic AD involves intracerebroventricular (ICV) injection with streptozotocin (STZ). However, ICV injections are an invasive approach, which creates limitations in generalizing the results. In this study, we produced a rodent model of AD using STZ (3 mg/kg) injection via the cisterna magna (CM) once every week for 4 weeks, and analyzed at 4 weeks and 16 weeks after final injection. In the CM-STZ rodent model of AD, we observed increase in extracellular amyloid-beta (Aβ) deposition and decrease and abnormal morphology of post-synaptic protein, PSD95 in 16 weeks STZ-injected group. The model developed using our less-invasive method induced features of AD-like pathology, including significantly increased extracellular amyloid-beta deposition, and decreased synaptic protein in the hippocampus. These findings supporting the success of this alternative approach, and thus, we suggest this is a promising, less invasive model for use in future AD research.
Revision of the life cycle of the harmful dinoflagellate Margalefidinium polykrikoides (Gymnodiniales, Dinophyceae) based on isolates from Korean coastal waters
No full textAlthough the life cycle of Margalefidinium polykrikoides, formerly called Cochlodinium polykrikoides, has been described previously, the descriptions on asexual and sexual phases of this species collected from Korean coastal waters are strange and still unclear. We collected the resting cysts of M. polykrikoides from surface sediments and re-examined the germination process, and asexual and sexual life stages, and the mating behavior of the vegetative cells using a microscope equipped with high-resolution time-lapse digital camera. The resting cyst of M. polykrikoides had a distinct germination process; before germination, a mature cell in shape was visible within the resting cyst body, and the cell quickly emerged from the resting cyst. The asexual reproduction and cell chain formation of M. polykrikoides were generated in a slightly oblique direction at the epicone of cell and the chain-forming cells of M. polykrikoides could form the chain-forming temporary cysts without asexual or sexual reproduction. The planozygote produced by gamete fusion of M. polykrikoides may not necessarily develop to resting cysts but instead may divide into vegetative cells occasionally. In this phase, the armored cell, which was previously recorded by Kim et al. (2007), was not observed. The sexuality of M. polykrikoides had a homothallic mating behavior.
Comparative studies of the expression of creatine kinase isoforms under immune stress in Pelodiscus sinensis
No full textThe expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis.
Cathelicidin-related antimicrobial peptide contributes to host immune responses against pulmonary infection with Acinetobacter baumannii in mice
No full textAcinetobacter baumannii is known for its multidrug antibiotic resistance. New approaches to treating drug-resistant bacterial infections are urgently required. Cathelicidin-related antimicrobial peptide (CRAMP) is a murine antimicrobial peptide that exerts diverse immune functions, including both direct bacterial cell killing and immunomodulatory effects. In this study, we sought to identify the role of CRAMP in the host immune response to multidrug-resistant Acinetobacter baumannii. Wild-type (WT) and CRAMP knockout mice were infected intranasally with the bacteria. CRAMP-/- mice exhibited increased bacterial colony-forming units (CFUs) in bronchoalveolar lavage (BAL) fluid after A. baumannii infection compared to WT mice. The loss of CRAMP expression resulted in a significant decrease in the recruitment of immune cells, primarily neutrophils. The levels of IL-6 and CXCL1 were lower, whereas the levels of IL-10 were significantly higher in the BAL fluid of CRAMP-/- mice compared to WT mice 1 day after infection. In an in vitro assay using thioglycollate-induced peritoneal neutrophils, the ability of bacterial phagocytosis and killing was impaired in CRAMP-/- neutrophils compared to the WT cells. CRAMP was also essential for the production of cytokines and chemokines in response to A. baumannii in neutrophils. In addition, the A. baumannii-induced inhibitor of κB-α degradation and phosphorylation of p38 MAPK were impaired in CRAMP-/- neutrophils, whereas ERK and JNK phosphorylation was upregulated. Our results indicate that CRAMP plays an important role in the host defense against pulmonary infection with A. baumannii by promoting the antibacterial activity of neutrophils and regulating the innate immune responses.
Lysobacter profundi sp. Nov., isolated from freshwater sediment and reclassification of Lysobacter panaciterrae as Luteimonas panaciterrae comb. nov.
No full textA polyphasic taxonomic study was carried out on strains CHu50b-3-2T and CHu40b-3-1 isolated from a 67 cm-long sediment core collected from the Daechung Reservoir at a water depth of 17 m, Daejeon, Republic of Korea. The cells of the strains were Gram-stain-negative, non-spore-forming, non-motile and rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of two strains with γ-Proteobacteria, which showed the highest pairwise sequence similarities to Lysobacter hankyongensis KTce-2T (96.5%), Lysobacter pocheonensis Gsoil193T (96.3%), Lysobacter ginsengisoli Gsoil 357T (96.1%), Lysobac-ter solanacearum T20R-70T (96.1%), Lysobacter brunescens KCTC 12130T (95.4%) and Lysobacter capsici YC5194T (95.3%). The phylogenetic analysis based on 16S rRNA gene sequences showed that the strains formed a clear phylogenetic lineage with the genus Lysobacter. The major fatty acids were identified as summed feature 9 (iso-C17: 1 ω9c and/or C18: 1 10-methyl), iso-C15: 0, iso-C16: 0 and iso-C17: 0. The respiratory quinone was identified as ubiquinone Q-8. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. The genomic DNA G+C content was determined to be 66.8 mol% (genome) for strain CHu50b-3-2T and 66.4 mol% (HPLC) for strain CHu40b-3-1. Based on the combined genotypic and phenotypic data, we propose that strains CHu50b-3-2T and CHu40b-3-1 represent a novel species of the genus Lysobacter, for which the name Lysobacter profundi sp. nov. is proposed. The type strain is CHu50b-3-2T (=KCTC 72973T=CCTCC AB 2019129T). Besides Lysobacter panaciterrae Gsoil 068T formed a phylogenetic group together with strain Luteimonas aquatica RIB1-20T (EF626688) that is clearly separated from all other known Lysobacter strains. Based on the phylogenetic relationships together with fatty acid compositions, Lysobacter panaciterrae Gsoil 068T should be reclassified as a member of the genus Lute-imonas: Luteimonas aquatica comb. nov. (type strain Gsoil 068T=KCTC 12601T=DSM 17927T).
Zwitterionic polydopamine/protein G coating for antibody immobilization: toward suppression of nonspecific binding in immunoassays
No full textFor the development of immunoassays into sophisticated analyte-sensing methods, it is a priority to suppress nonspecific binding in immunoassays. Herein, we report a one-step surface coating method that can not only optimally immobilize antibodies but also suppress nonspecific binding. Zwitterionic dopamine (ZW-DOPA) exhibits distinct antifouling performance, and protein G enables an antibody to have an optimal orientation. A mixture of ZW-DOPA and protein G can be simply coated onto various kinds of surfaces, and the antibody can be immobilized onto the ZW-DOPA/protein G-coated surfaces. The antifouling property of the zwitterionic group, surface-independent coating property of the catechol and amine groups, and antibody-retaining property of protein G synergistically contribute to surface-independent and oriented immobilization of antibodies without nonspecific binding. The surface characteristics of ZW-DOPA/protein G-coated substrates were analyzed by X-ray photoelectron spectroscopy, contact angle goniometry, atomic force microscopy, and ellipsometry. Importantly, the ZW-DOPA/protein G-coated substrates showed high resistance to nonspecific protein adhesion. We also verified that antibodies could be immobilized onto ZW-DOPA/protein G-coated substrates using fluorescence and biolayer interferometry systems. Finally, ZW-DOPA/protein G-coated substrates were employed as immune substrates for influenza virus detection via the naked eye and surface-enhanced Raman scattering, allowing us to efficiently identify the virus. It is anticipated that the developed ZW-DOPA/protein G coating method will be useful for the advancement of immunoassays.