Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.Aptamers are artificially selected oligonucleotides that bind to molecular targets, typically proteins, with high specificity and avidity (1–3). DNA aptamers have been selected against dozens of targets for biomedical applications both as therapeutics (4, 5) and diagnostics (6, 7). Despite their widespread application, few DNA aptamer-target complex structures have been solved (8)–the best studied of which is the G-quadruplex aptamer that binds to thrombin (9–12). A DNA aptamer that binds to von Willebrand factor showed a three-stem structure of mainly B-form DNA with some noncanonical base pairing (13). Most recently, the structure of an innovative Slow Off-rate Modified Aptamer (SOMAmer) bound to platelet-derived growth factor B was solved, revealing binding via a hydrophobic surface that mimics how the factor binds to its receptor (14). Generally, the lack of DNA aptamer-target structures has limited our understanding of the mechanisms by which DNA aptamers attain their specificity (15), resulting in a bias in aptasensor development (16).Better point-of-care tests are critically needed for malaria, a disease which continues to claim more than 1 million lives globally every year (17). Antimalarial drugs have been administered presumptively to patients with fever for decades, leading to drug resistance and poor management of other febrile illness. The cost of newer, more effective treatments has led to a situation whereby improved diagnostics has become a major factor that could reduce the burden of malaria in the developing world (17). Antibody-based rapid diagnostic tests have greatly benefitted malaria management, but significant issues with cost (17) and stability in tropical climates (18) remain that are intrinsically associated with the use of protein antibodies. DNA aptamers compare favorably to antibodies for diagnostic applications (19) with particular advantages that could be critical for diagnostic tests of the developing world: thermal stability, convenient chemical synthesis, and potentially lower costs of production (16). Here, we report the crystal structure and application of a unique DNA aptamer against an established malaria pan-species diagnostic target, Plasmodium falciparum lactate dehydrogenase (PfLDH) (20), and a mechanism of molecular recognition by a distorted hairpin DNA aptamer.     

Defining criteria for the introduction of liraglutide using the glucagon stimulation test in patients with type 2 diabetes

Aims/Introduction

To define a set of criteria using indices of β‐cell function, including results from the glucagon stimulation test, for liraglutide introduction in patients with type 2 diabetes.

Materials and Methods

In the present retrospective cohort study, patients were included in our analysis if their β‐cell function had been evaluated with a glucagon stimulation test and a 24‐h urinary C‐peptide (U‐CPR) excretion test before switching from insulin therapy to liraglutide monotherapy. The efficacy of liraglutide was determined by the extent to which glycemic control was achieved or if glycated hemoglobin levels were maintained at <7.0% after liraglutide monotherapy for 24 weeks.

Results

Liraglutide was effective in 36 of 77 patients. In the liraglutide‐effective cases, the following parameters were higher: fasting C‐peptide (CPR0) levels, C‐peptide levels 6 min after glucagon stimulation (CPR6), the C‐peptide index (CPI; CPR0 × 100/fasting plasma glucose) and stimulated C‐peptide index (S‐CPI; CPR6 × 100/plasma glucose 6 min after glucagon stimulation). U‐CPR did not differ between liraglutide‐effective and liraglutide‐ineffective cases. Using receiver operating characteristic analysis adjusted for baseline characteristics, the independent cut‐off value for effective liraglutide introduction was 0.72 for CPI and 1.92 for S‐CPI.

Conclusions

Evaluation of β‐cell function using the glucagon stimulation test is useful for determining the efficacy of liraglutide introduction in patients with type 2 diabetes.     

Crizotinib administered via nasogastric and percutaneous endoscopic gastrostomy tubes for the successful treatment of ALK-rearranged lung cancer in a patient with poor performance status

Crizotinib—an anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor—is effective in non-small-cell lung cancers (NSCLCs) that express ALK. Here, we report a patient with ALK-positive lung adenocarcinoma who was administered crizotinib via nasogastric and percutaneous endoscopic gastrostomy (PEG) tubes, with positive results. This case indicates that patients with ALK-positive NSCLC may successfully be treated with crizotinib via nasogastric or PEG tubes. This approach can even be used as a salvage treatment in patients with poor prognoses.     

Über die Kombination angeborener und erworbener Skeletterkrankungen

Ohne ZusammenfassungMit 16 Textfiguren und 3 Tafeln.     

Collagen metabolism following corneal laceration in rabbits

We investigated the synthesis and degradation of collagen in lacerated rabbit corneas. The rate of collagen synthesis was measured by the incorporation and hydroxylation of [14C]proline up to 5 weeks after a penetrating laceration. Our results indicate that protein and collagen synthesis is initially reduced during the 24 h period after laceration and then increases in two phases. A moderate increase in protein and collagen synthesis occurs up to day 5 after laceration. Synthesis then decreases to a low level through the 10th day after injury. A second wave of increase in protein and collagen synthesis takes place reaching a new peak of approximately twice the activity as found in control corneas after 5 weeks of healing. Afterwards, the rate of protein and collagen synthesis declines and reaches the basal level after 7 weeks of healing. In a separate set of experiments, rabbit corneas were lacerated and allowed to heal for one or three weeks at which point 100 microCi of [14C]proline was injected into each anterior chamber. The corneas were excised 1 to 17 days later and subjected to collagen analysis. Our data indicates that the degradation of collagen synthesized by the cornea 1 week after laceration followed biphasic kinetics. In the first phase, the half-life of newly-synthesized collagen is 20 days, whereas it is approximately 4 days in the second phase. It is of particular interest that the accelerated degradation of the newly-synthesized [14C]collagen is concomitant with the increased rate of collagen synthesis during the wound healing process. The degradation of collagen synthesized 3 weeks after injury was slower and followed monophasic kinetics having a half-life of 14 days. The degradation of non-collagenous 14C-proteins follow a monophasic kinetics having a half-life of 6-7 days. Polyacrylamide gel electrophoresis of newly-synthesized collagen indicated that the collagen(I) and collagen(V) were the main collagenous components synthesized by the lacerated corneas.     

Poly(Ethyleneimine)-Immobilized-Cloth Enzyme Immunoassay for the Detection of Salmonella Lipopolysaccharide

Poly(ethyleneimine) (PEI) was immobilized on non-woven polyester cloth and examined for application on a simple, rapid and economical “cloth enzyme immunoassay (CEIA)” which was developed originally as polymyxin-CEIA for the detection of Salmonella lipopolysaccharide (LPS). PEI-cloth regardless of the PEI molecular weight, but with the amine group contents of 0.1 ∼ 0.35 meq/g immobilized either in a physisorption-like or chemisorption-like manner, adsorbed LPS rapidly, preferentially and effectively. The captured LPS was then able to be detected qualitatively and quantitatively as an antigen by enzyme immunoassay. PEI-CEIA had a detection limit for Salmonella LPS of 10 ng/ml, which was equivalent to 1.6 × 10⁵ cell/ml and was ten times more sensitive than polymyxin-CEIA. It was possible to detect Salmonella LPS in the presence of a 100-fold excess of E. coli LPS. PEI-CEIA was found to be more sensitive and much easier to carry out than polymyxin-CEIA but had the same advantages as polymyxin-CEIA.     

Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant.

BACKGROUND: We previously reported that puromycin aminonucleoside (PAN) increased adrenomedullin (AM) secretion and AM mRNA expression in podocytes, through overproduction of oxidative stress. To clarify the cytoprotective role of AM as antioxidative and antiapoptotic substance in podocytes, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis in conditionally immortalized murine podocytes. METHODS: The expression of AM, RAMP 2 and RAMP 3 was investigated using real-time PCR, western blotting analysis and immunofluorescence microscopy. Reactive oxygen species (ROS) production was measured by CM-H(2)DCFDA fluorescence intensity method. The percentage of apoptotic cells was measured by Hoechst 33342 staining. RESULTS: PAN (100 microg/ml) significantly (P < 0.01) increased ROS production, associated with an increase in apoptosis; the percentage of apoptotic cells is 5.3% + 0.05% (P < 0.01) with 36 h treatment of PAN compared to 0.24 + 0.16% with no treatment. Several antioxidants could markedly reduce PAN-induced apoptosis in cultured podocytes, suggesting that PAN-induced apoptosis might be attributable to the overproduction of ROS. Accordingly, the administration of exogenous AM (10(-6) M) could significantly reduce not only ROS production via a PKA-dependent pathway, but also the resultant apoptosis induced by PAN. AM antagonists, CGRP8-37, augmented PAN-induced apoptosis, associated with increased ROS production, 2.2- and 2.3-Fold, respectively. RAMP 2 and RAMP 3 could be detected in podocytes and glomeruli. CONCLUSIONS: This suggests that ROS-induced up-regulation of AM with PAN could counteract ROS-induced apoptosis, by the suppression of ROS production. Therefore, AM might have the endogenous antioxidant potential to protect against ROS-induced podocyte injury.