Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

Facile generation of biepitopic antibodies with intrinsic agonism for activating receptors in the tumor necrosis factor superfamily.

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.

Detection of a novel porcine circovirus 4 in Korean pig herds using a loop-mediated isothermal amplification assay.

A novel porcine circovirus 4 has been recently identified in China and Korea. A sensitive and specific diagnostic method is urgently required to detect the virus in field samples. We developed a loop-mediated isothermal amplification (LAMP) the assay for the visual detection of PCV4 and evaluated its sensitivity, specificity, and applicability in clinical samples. This assay's results can be directly visualized by the naked eye using hydroxynaphthol blue after incubation for 40 min at 64 °C. The assay specifically amplified PCV4 DNA and no other viral nucleic acids. The sensitivity of the assay was <50 DNA copies/reaction, which was 10 times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). Clinical evaluation revealed that the PCV4 detection rate in individual pig samples and at the farm level was 39.3 % (57/145) and 45.7 % (32/70), respectively, which were higher than cPCR (46 samples, 24 farms) and qPCR (52 samples, 29 farms) results. Cumulatively, owing to the advantages of high sensitivity and specificity, direct visual monitoring of the results, no possibility for cross-contamination, and being a low-cost equipment, the developed LAMP assay will be a valuable tool for the detection of the novel PCV4 in clinical samples, even in resource-limited laboratories.

A simple colorimetric detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification assay using hydroxynaphthol blue metal indicator.

A simple reverse transcription loop-mediated isothermal amplification combined with visual detection method (vRT-LAMP) assay was developed for rapid and specific detection of porcine epidemic diarrhea virus (PEDV) in this study, which overcomes the shortcomings of previously described RT-LAMP assays that require additional detection steps or pose a risk of cross-contamination. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubating for 40 min at 62 °C. The assay specifically amplified PEDV RNA and no other viral nucleic acids. The limit of detection of the assay was less than 50 RNA copies per reaction, which was 100 times more sensitive than conventional reverse transcription polymerase chain reaction (RT-PCR) and comparable to real-time RT-PCR (RRT-PCR). In the clinical evaluation, the PEDV detection rate of vRT-LAMP was higher than that of RRT-PCR, showing 99 % concordance, with a kappa value (95 % confidence interval) of 0.97 (0.93-1.01). Considering the advantages of high sensitivity and specificity, simple and direct visual monitoring of the results, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vRT-LAMP assay will be a valuable tool for detecting PEDV from clinical samples, even in resource-limited laboratories.

The application of helix fusion methods in structural biology.

Methods generating fusion proteins with rigid and predictable structures have been developed in recent years. Among them, helix fusion methods that link two proteins by connecting their terminal alpha helices into a single and extended alpha helix can be particularly useful because designing fusion helices is conceptually and technically simple. These methods have been shown crucial in obtaining crystals that diffract x-rays to high resolution or attaching large and symmetrical backbone proteins to small target proteins for cryo-EM analysis. The structural rigidity of the fusion helix is crucial for these applications, and the reduction of structural ambiguity and flexibility at the fusion sites will further enhance the usefulness of this method.

Structural diversity and flexibility of diabodies.

Diabodies are bispecific antibody fragments that have two antigen binding Fv domains. They are unique among hundreds of different formats of bispecific antibodies because they are small and rigid enough to be crystallized. Diabodies are generated by connecting variable regions of heavy and light chains by a peptide linker. Because of the short length of the linker, intramolecular association of the variable regions is not allowed. Instead, the variable regions from the different peptide chains associate together, forming a dimeric complex with two antigen binding sites. Previous crystallographic studies of diabodies demonstrate the extraordinary structural diversity of diabodies. They have also shown that the relative orientation and interaction of the two Fv domains in diabodies have substantial flexibility due to instability of the Fv interface. Introduction of site specific mutations and disulfide bridges can reduce flexibility and therefore increase rigidity and predictability of the diabody structures. These stabilized diabodies will be useful for future application to structural biology and protein nanotechnology.

Construction of novel repeat proteins with rigid and predictable structures using a shared helix method.

Generating artificial protein assemblies with complex shapes requires a method for connecting protein components with stable and predictable structures. Currently available methods for creating rigid protein assemblies rely on either complicated calculations or extensive trial and error. We describe a simple and efficient method for connecting two proteins via a fused alpha helix that is formed by joining two preexisting helices into a single extended helix. Because the end-to-end ligation of helices does not guarantee the formation of a continuous helix, we superimposed 1-2 turns of pairs of connecting helices by using a molecular graphics program. Then, we chose amino acids from the two natural sequences that would stabilize the connecting helix. This "shared helix method" is highly efficient. All the designed proteins that could be produced in Escherichia coli were readily crystallized and had the expected fusion structures. To prove the usefulness of this method, we produced two novel repeat proteins by assembling several copies of natural or artificial proteins with alpha helices at both termini. Their crystal structures demonstrated the successful assembly of the repeating units with the intended curved shapes. We propose that this method could dramatically expand the available repertoire of natural repeat proteins.

Efficacy and safety of evogliptin monotherapy in patients with type 2 diabetes and moderately elevated glycated haemoglobin levels after diet and exercise.

AIMS: To evaluate the efficacy and safety of evogliptin, a newly developed dipeptidyl peptidase-4 inhibitor, in patients with type 2 diabetes (T2D) inadequately controlled by diet and exercise. MATERIALS AND METHODS: In this randomized, double-blind, placebo-controlled, parallel-group, multicentre, phase III study, 160 patients with T2D were assigned to either evogliptin 5 mg or placebo for 24 weeks. The primary endpoint was the mean change in glycated haemoglobin (HbA1c) from baseline to week 24. RESULTS: The mean baseline HbA1c levels were similar in the evogliptin and the placebo groups (7.20% ± 0.56% vs 7.20% ± 0.63%, respectively). At week 24, evogliptin significantly reduced HbA1c levels from baseline compared with placebo (-0.23% vs 0.05%, respectively, P < .0001). Additionally, the proportion of patients achieving HbA1c <6.5% was significantly higher in the evogliptin group than in the placebo group (33.3% vs 15.2%; P = .008). The overall incidence of adverse events, including hypoglycaemia, was similar in the 2 groups. CONCLUSIONS: In this 24-week study, once-daily evogliptin monotherapy significantly improved glycaemic control and was well tolerated in patients with T2D.

Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress.

BACKGROUND: Clonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme. METHODS: We isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm's reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated. RESULTS: The full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8-44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing. CONCLUSIONS: CsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.

Highly selective cysteine detection and bioimaging in zebrafish through emission color change of water-soluble conjugated polymer-based assay complex.

A new concept for rapid, label-free cysteine sensing method is proposed via possible naked eye-detection of red-to-blue emission color change. Intermolecular exciton migration in conjugated polyelectrolyte-based assay complex is adopted to enhance selectivity and sensitivity for cysteine sensing by formation and dissociation of polymer-Hg(2+)-thymine assay complex in the absence and presence of cysteine, respectively. The assay complex shows red emission due to cooperative aggregation of conjugated polyelectrolyte, thymine, and Hg(2+). Upon exposure to cysteine, the assay complex dissociates into individual molecules showing transparent, blue-emitting solution, because cysteine extracts Hg(2+) from the assay complex via more favorable binding between cysteine and Hg(2+).

Simultaneous detection and removal of mercury ions in aqueous solution with fluorescent conjugated polymer-based sensor ensemble.

A water-soluble, sulfur-containing fluorescent conjugated polymer exhibits a visible fluorescence color change for detection of mercury in the presence of thymine. A new concept provides the design of a sensor ensemble using a simple combination method. This strategy avoids the need for complicated design and synthesis of a recognition group, eliminating the tedious synthetic efforts for the preparation of a sensor material.

Self-assembly of supramolecualr metallogelator containing 2-(2'-hydroxyphenyl) benzoxazole/Zn(II) chelate.

A low molar mass organogelator 1 containing 2-(2'-hydroxyphenyl)benzoxazole (HPB) unit with long alkyl chain was synthesized by the reaction with HPB and octyl isocyanate in THF at room temperature. A new chelate-based organogelator 1-Zn(II) was prepared with the reaction of 1 and zinc(II) acetate in methanol and dichloromethane at mild condition. The gelation ability of organogelator was tested by heating-cooling method in various organic solvents, and the opaque gel was formed from DMF. Well-developed self-assembled structure of organogel was confirmed with field emission-scanning electron microscope (FE-SEM) and transmission electron microscope (TEM), and the optical properties of organogel upon aggregation were monitored by UV-Vis and fluorescence spectroscopy, 1-Zn(II) was self-assembled to the sheet-like structure with the thickness of fully extended length of molecules.

A Web-based mobile asthma management system.

Continuous recording of daily symptoms constitutes an effective means of managing asthma patients. Daily management reduces the costs associated with hospitalization and improves the quality of patient care. We have implemented a Web-based mobile asthma management system. We used a pocket PC, mobile phone and desktop computer. The recorded items and individualized prescriptions were structured using Extensible Mark-up Language (XML) DTD (Data Type Definition). The mobile Web form was automatically adjusted to fit the different display resolutions of the terminal devices. The system provided reliable exchange of all relevant information between a doctor and the asthma patient using wireless mobile transmission. E-mail and Short Messaging Service (SMS) were used to send messages to patients, for example in the case of an automatically determined patient alert. Patients could obtain customized instructions according to their daily personal symptoms, peak expiratory flow (PEF), medications and activity restriction. The daily graph of PEF and the graphs of symptoms and medication were particularly useful for asthma patient control and self-awareness of the progress of the disease.

Do acute changes in heart rate by isoproterenol affect aortic stiffness in patients with hypertension?

BACKGROUND: Increased aortic stiffness is a independent risk factor of cardiovascular disease in patients with hypertension. Acute changes of the heart rate (HR) have been reported not to affect the aortic stiffness in pacing. However, it is unknown whether acute changes in HR caused by sympathomimetics can affect the aortic stiffness in patients with hypertension. We investigated the effect of acute changes in HR produced by isoproterenol on the aortic stiffness in 17 hypertensive patientss (mean age: 59 +/- 9 years). METHODS: All vasoactive drugs were discontinued at least 3 days before the study. The carotid-to-femoral pulse wave velocity (PWV) was measured by the foot-to-foot method. The pulse waves were recorded at the baseline and at every increase of HR by 5 to 10 bpm with a gradual increase of the dose of isoproterenol. The blood pressures and HR were measured simultaneously. For the analysis, HR, PWV, compliance (C), and compliance index (Ci) were converted as percent changes (delta) from the baseline values. Percent changes of the parameters of the aortic stiffness, i.e., deltaPWV, deltaC, and deltaCi, were grouped by every 10% increase in deltaHR. RESULTS: There was no significant difference among groups in deltaPWV, deltaC and deltaCi (p > 0.05 for each of the group). The regression analysis showed no significant correlation of deltaHR with deltaPWV and deltaC (r = 0.18, 0.13 respectively, p > 0.05 for each). deltaCi had a poor correlation with deltaHR (r = 0.22, p < 0.05). However, only 4.6% of deltaCi could be referred to deltaHR (r2 = 0.046). CONCLUSION: Aortic stiffness was not affected by acute changes in HR produced by isoproterenol which suggests that it is not necessary to consider acute changes in HR when measuring aortic PWV.

The resistance mechanisms of b-lactam antimicrobials in clinical isolates of Acinetobacter baumannii.

BACKGROUND: Despite increasing importance of Acinetobacter baumannii in nosocomial infections and rapid development of multi-antimicrobial resistance in this strain, the resistance mechanisms of beta-lactam antimicrobials in A. baumannii were not clearly defined. In order to observe the resistance mechanisms against beta-lactams and carbapenem, we characterized the production of beta-lactamases and outermembrane protein (OMP) profiles for the 44 clinical isolates of A. baumannii. METHODS: The MICs of antimicrobials were determined by agar dilution test. The secondary beta-lactamases were characterized by isoelectric focusing, polymerase chain reactions and nucleotide sequencing, and the production of chromosomal beta-lactamases was quantitated by spectrophotometric method. For two strains with an elevated MIC of carbapenem, outermembrane protein (OMP) profile was analyzed by ultracentrifugation of the sonicated bacteral cells and SDS-PAGE. RESULTS AND CONCLUSION: Twenty two or 4 of 44 strains produced TEM-1-like beta-lactamase or PER-1 extended-spectrum beta-lactamase, respectively. However, when we analyzed the MICs of several beta-lactams with the beta-lactamase production, the resistance level of beta-lactam was mainly determined by the production of chromosomal beta-lactamase, not by the secondary beta-lactamases in the clinical isolates of A. baumannii. In two strains with an elevated MIC of imipenem, a decrease or loss of about 35 kDa and 22 kDa proteins in OMP was observed, which suggested that the change of OMP played a role in carbapenem resistance.