Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.     

Achene morphology of Saussurea species (Asteraceae,Cardueae) in Korea and its systematic implications

Achene size and shape, surface sculpturing, and pericarp and testa wall structure of 23 Korean Saussurea spp. were investigated using scanning electron microscopy (SEM) and light microscopy to evaluate the infrageneric relationships and assess their systematic significance. Achene size categories and thickness of the testa epidermis were distinguished using biometric measurements. Four basic types of surface pattern were observed: (1) lineate; (2) striate; (3) reticulate; and (4) colliculate. Saussurea rorinsanensis was found to have some unique achene characteristics, such as a fusiform achene, uniform pappus, presence of epidermal hairs and tangentially elongated, narrow testa epidermal cells. The characteristic achene features for species were found to be achene size and shape, hilum position, surface sculpture, pappus composition, morphology of the pericarp wall and thickness of the testa epidermis. Based on 16 morphological and achene characters, a cladistic analysis resolved three well‐supported clades, with S. eriophylla as the first‐branching taxon. Saussurea pulchella and S. japonica, both belonging to Saussurea subgenus Theodorea, were distant from each other in the 50% majority rule consensus tree and the character distribution cladogram. This cladistic analysis of achene morphology and anatomy should be regarded as giving us a tentative picture of the phylogenetics of Saussurea, and this study may serve as a reference for future hypotheses and studies on the characterization and classification of Saussurea spp. in Korea.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver Organoids

1. Download : Download high-res image (187KB)
2. Download : Download full-size image

     

Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide

TLR2 in association with TLR1 or TLR6 plays an important role in the innate immune response by recognizing microbial lipoproteins and lipopeptides. Here we present the crystal structures of the human TLR1-TLR2-lipopeptide complex and of the mouse TLR2-lipopeptide complex. Binding of the tri-acylated lipopeptide, Pam(3)CSK(4), induced the formation of an "m" shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam(2)CSK(4), did not. The three lipid chains of Pam(3)CSK(4) mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR1-TLR2 heterodimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.     

Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells

A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.     

A simple fluorescent method for detecting mismatched DNAs using a MutS-fluorophore conjugate

A fluorescent method was developed for the detection of unpaired and mismatched DNAs using a MutS-fluorophore conjugate. The fluorophore, 2-(4'-(iodoacetoamido)anilino) naphthalene-6-sulfonic acid (IAANS), was site-specifically attached to the 469 position of Thermus aquaticus (Taq.) MutS mutant (C42A/T469C). The fluorophore labeled residue located at the dimer interface of the protein undergoes a drastic conformational change upon binding with mismatched DNA. The close proximity of the two identical fluorescent molecules presumably causes the self-quenching of the fluorophore, since fluorescence emission of the biosensor decreases with increasing concentrations of mismatched DNA. The order of binding affinity for each unpaired and mismatched DNA obtained by this method was DeltaT (Kd=52 nM)>GT (62 nM)>DeltaC (130 nM)>CT (160 nM)>DeltaG (170 nM)>DeltaA (250 nM)>CC (720 nM)>AT (950 nM). This order is comparable to the previous results of the gel mobility shift assay. Thus, this method can be a simple, useful tool for elucidating the mechanism of DNA mismatch repair as well as a novel probe for detecting of genetic mutation.