Visualizing reactive astrocyte-neuron interaction in Alzheimer's disease using 11C-acetate and 18F-FDG.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.

Amyloid Against Amyloid: Dimeric Amyloid Fragment Ameliorates Cognitive Impairments by Direct Clearance of Oligomers and Plaques.

Amyloid-ß (Aß) in the form of neurotoxic aggregates is regarded as the main pathological initiator and key therapeutic target of Alzheimer's disease. However, anti-Aß drug development has been impeded by the lack of a target needed for structure-based drug design and low permeability of the blood-brain barrier (BBB). An attractive therapeutic strategy is the development of amyloid-based anti-Aß peptidomimetics that exploit the self-assembling nature of Aß and penetrate the BBB. Herein, we designed a dimeric peptide drug candidate based on the N-terminal fragment of Aß, DAB, found to cross the BBB and solubilize Aß oligomers and fibrils. Administration of DAB reduced amyloid burden in 5XFAD mice, and downregulated neuroinflammation and prevented memory impairment in the Y-maze test. Peptide mapping assays and molecular docking studies were utilized to elucidate DAB-Aß interaction. To further understand the active regions of DAB, we assessed the dissociative activity of DAB with sequence modifications.

Prediction of pharmacokinetics and drug-drug interaction potential using physiologically based pharmacokinetic (PBPK) modeling approach: A case study of caffeine and ciprofloxacin.

Over the last decade, physiologically based pharmacokinetics (PBPK) application has been extended significantly not only to predicting preclinical/human PK but also to evaluating the drug-drug interaction (DDI) liability at the drug discovery or development stage. Herein, we describe a case study to illustrate the use of PBPK approach in predicting human PK as well as DDI using in silico, in vivo and in vitro derived parameters. This case was composed of five steps such as: simulation, verification, understanding of parameter sensitivity, optimization of the parameter and final evaluation. Caffeine and ciprofloxacin were used as tool compounds to demonstrate the "fit for purpose" application of PBPK modeling and simulation for this study. Compared to caffeine, the PBPK modeling for ciprofloxacin was challenging due to several factors including solubility, permeability, clearance and tissue distribution etc. Therefore, intensive parameter sensitivity analysis (PSA) was conducted to optimize the PBPK model for ciprofloxacin. Overall, the increase in Cmax of caffeine by ciprofloxacin was not significant. However, the increase in AUC was observed and was proportional to the administered dose of ciprofloxacin. The predicted DDI and PK results were comparable to observed clinical data published in the literatures. This approach would be helpful in identifying potential key factors that could lead to significant impact on PBPK modeling and simulation for challenging compounds.

Aptamer selection for fishing of palladium ion using graphene oxide-adsorbed nanoparticles.

A new aptamer selection method using graphene oxide (GO)-adsorbed nanoparticles (GO-adsorbed NPs) was employed for specific fishing of palladium ion. High affinity ssDNA aptamers were isolated through 13 rounds of selection and the capacity of the selected DNA aptamers for palladium ion uptake was measured, clarifying that DNA01 exhibits the highest affinity to palladium ion with a dissociation constant (Kd) of 4.60±1.17 µM. In addition, binding ability of DNA01 to palladium ion was verified against other metal ions, such as Li(+), Cs(+), Mg(2+), and Pt(2+). Results of the present study suggest that future modification of DNA01 may improve palladium ion-binding ability, leading to economic recovery of palladium from water solution.

TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3.

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5'-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with V(max) of 41.4±3.3 nM/min and K(M) of 1.60±0.23 µM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.

A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay.

A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3',5'-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.

Understanding the pharmacokinetic journey of Fc-fusion protein, rhIL-7-hyFc using complementary approach of two analytical methods, accelerator mass spectrometry and ELISA.

Antibody-based therapeutics (ABTs), including monoclonal/polyclonal antibodies and fragment crystallizable region (Fc)-fusion proteins, are increasingly used in disease treatment, driving the global market growth. Understanding the pharmacokinetic (PK) properties of ABTs is crucial for their clinical effectiveness. This study investigated the PK profile and tissue distribution of efineptakin alfa, a long-acting recombinant human interleukin-7 (rhIL-7-hyFc), using enzyme-linked immunosorbent assay (ELISA) and accelerator mass spectrometry (AMS). Totally, four rats were injected intramuscularly with 1 mg/kg of rhIL-7-hyFc containing 14C-rhIL-7-hyFc, which was prepared via reductive methylation. Serum total radioactivity (TRA) and serum rhIL-7-hyFc concentrations were quantified using AMS and ELISA, respectively. The TRA concentrations in organs were determined by AMS. Serum TRA peaked at 10 hours with a terminal half-life of 40 hours. The rhIL-7-hyFc exhibited a mean peak concentration at around 17 hours and a rapid elimination with a half-life of 12.3 hours. Peak concentration and area under the curve of TRA were higher than those of rhIL-7-hyFc. Tissue distribution analysis showed an elevated TRA concentrations in lymph nodes, kidneys, and spleen, indicating rhIL-7-hyFc's affinity for these organs. The study also simulated the positions of 14C labeling in rhIL-7-hyFc, identifying specific residues in the fragment of rhIL-7 portion, and provided the explanation of distinct analytes targeted by each method. Combining ELISA and AMS provided advantages by offering sensitivity and specificity for quantification as well as enabling the identification of analyte forms. The integrated use of ELISA and AMS offers valuable insights for the development and optimization of ABT.

Environmental forensics using stable and radioactive isotopes in naturally attenuated soil after phenol-leakage accidents.

Phenol is a carcinogenic and hazardous chemical used in multiple industries and poses a high risk of chemical spills into the environment. To date, environmental forensic research has not focused on chemically remediated soils. In this study, an advanced environmental forensic analysis was performed on microbial communities and breakdown products of phenol, carbon stable isotopes, and radioactive isotopes in phenol-contaminated soil. As indicators of phenol-spill accidents after natural attenuation, higher δ13C levels and lower 14C/12C ratios were observed in phenol-contaminated soil compared with uncontaminated soil. In addition, 16s rRNA gene analysis revealed that phenol-breakdown products identified by gas chromatography-mass spectrometry and the presence of soil bacteria, such as Nocardioides, Faecalibacterium, and Bacteroides, were indicators of phenol-leakage accidents. Therefore, the proposed environmental forensic strategy is a valuable tool for identifying the location of previously occurring chemical accidents and estimating the ecological impact after the natural attenuation of contaminated soils.

Verification of Therapeutic Effect through Accelerator Mass Spectrometry-Based Single Cell Level Quantification of hESC-Endothelial Cells Distributed into an Ischemic Model.

As the potential of pluripotent stem cell-derived differentiated cells has been demonstrated in regenerative medicine, differentiated vascular endothelial cells (ECs) are emerging as a therapeutic agent for the cardiovascular system. To verify the therapeutic efficacy of differentiated ECs in an ischemic model, human embryonic stem cells (hESCs) are induced as EC lineage and produce high-purity ECs through fluorescence-activated cell sorting (FACS). When hESC-ECs are transplanted into a hindlimb ischemic model, it is confirmed that blood flow and muscle regeneration are further improved by creating new blood vessels together with autologous ECs than the primary cell as cord blood endothelial progenitor cells (CB-EPCs). In addition, previously reported studies show the detection of transplanted cells engrafted in blood vessels through various tracking methods, but fail to provide accurate quantitative values over time. In this study, it is demonstrated that hESC-ECs are engrafted approximately sevenfold more than CB-EPCs by using an accelerator mass spectrometry (AMS)-based cell tracking technology that can perform quantification at the single cell level. An accurate quantification index is suggested. It has never been reported in in vivo kinetics of hESC-ECs that can act as therapeutic agents.

Levosimendan inhibits disulfide tau oligomerization and ameliorates tau pathology in TauP301L-BiFC mice.

Tau oligomers play critical roles in tau pathology and are responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization has become an important therapeutic strategy to treat tauopathies, including Alzheimer's disease. However, progress has been slow because detecting tau oligomers in the cellular context is difficult. Working toward tau-targeted drug discovery, our group has developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened libraries with FDA-approved and passed phase I drugs and identified levosimendan as a potent anti-tau agent that inhibits tau oligomerization. 14C-isotope labeling of levosimendan revealed that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan disassembles tau oligomers into monomers, rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents methylene blue and LMTM failed to protect neurons from tau-mediated toxicity, generating high-molecular-weight tau oligomers. Levosimendan displayed robust potency against tau oligomerization and rescued cognitive declines induced by tauopathy in the TauP301L-BiFC mouse model. Our data present the potential of levosimendan as a disease-modifying drug for tauopathies.

Coomassie blue is sufficient for specific protein detection of aptamer-conjugated chips.

An aptamer-based biochip for protein detection and quantitation which combines the recent biochip technology and the conventional staining methods, is described. Using a model system comprising His-tagged proteins as the analyte and single-stranded RNA aptamers specific for His-tagged proteins as immobilized ligands on chips, we could demonstrate that aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. The sensor has the characteristics of good stability, reproducibility and reusability, with detection limit as low as 85 ng/mL His-tagged protein. It has been demonstrated that the sensor can be stored for at least 4 weeks and reused with reasonable reduction rate of staining intensity. In conclusion, we could show the suitability of nucleic acid aptamers as low molecular weight receptors on biochips for sensitive and specific protein detection and quantitation.

In vivo tracking of 14C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry.

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.

Nanotracing and cavity-ring down spectroscopy: A new ultrasensitive approach in large molecule drug disposition studies.

New therapeutic biological entities such as bispecific antibodies targeting tissue or specific cell populations form an increasingly important part of the drug development portfolio. However, these biopharmaceutical agents bear the risk of extensive target-mediated drug disposition or atypical pharmacokinetic properties as compared to canonical antibodies. Pharmacokinetics and bio-distribution studies become therefore more and more important during lead optimization. Biologics present, however, greater analytical challenges than small molecule drugs due to the mass and selectivity limitation of mass spectrometry and ligand-binding assay, respectively. Radiocarbon (14C) and its detection methods, such as the emerging 14C cavity ring down spectroscopy (CRDS), thus can play an important role in the large molecule quantitation where a 14C-tag is covalently bound through a stable linker. CRDS has the advantage of a simplified sample preparation and introduction system as compared to accelerator mass spectrometry (AMS) and can be accommodated within an ordinary research laboratory. In this study, we report on the labeling of an anti-IL17 IgG1 model antibody with 14C propionate tag and its detection by CRDS using it as nanotracer (2.1 nCi or 77.7 Bq blended with the therapeutic dose) in a pharmacokinetics study in a preclinical species. We compare these data to data generated by AMS in parallel processed samples. The derived concentration time profiles for anti-IL17 by CRDS were in concordance with the ones derived by AMS and γ-counting of an 125I-labeled anti-IL17 radiotracer and were well described by a 2-compartment population pharmacokinetic model. In addition, antibody tissue distribution coefficients for anti-IL17 were determined by CRDS, which proved to be a direct and sensitive measurement of the extravascular tissue concentration of the antibody when tissue perfusion was applied. Thus, this proof-of-concept study demonstrates that trace 14C-radiolabels and CRDS are an ultrasensitive approach in (pre)clinical pharmacokinetics and bio-distribution studies of new therapeutic entities.

Discrimination of Lycium chinense and Lycium barbarum by taste pattern and betaine analysis.

Lycii Fructus was used as natural products with therapeutic properties for a long time. Betaine is a natural amino acid and one of the major constituents of Lycii Fructus. It is reported that this fruit plays a role in reducing blood levels of homocysteine, a toxic byproduct of the amino acid metabolism. This study was used to establish infra based on oriental medicine through the analysis of correlation of taste, contents of betaine, %Brix and physico-chemical properties of Lycii Fructus. To investigate betaine, quantitative analysis was performed using HPLC separation system. In addition, %Brix and saccharide were estimated. Taste pattern analysis was measured using the taste sensing system, SA402B equipped with six taste sensors including newly developed sweetness sensor. Betaine quantitative analysis showed that L. barbarum 0.64 ± 0.15% (n = 6) was significantly higher than L. chinense 0.55 ± 0.1% (n = 12). And %Brix and saccharide composition of Lycii Fructus analysis showed that L. barbarum was significantly higher than L. chinense. The results of taste pattern analysis between L. barbarum and L. chinense showed a significant difference in almost every taste. In contrast, sweetness of L. barbarum was higher than L. chinense. When clustering with sweetness and bitterness, the two species are distinctly separated. In conclusion, these taste patterns, %Brix, betaine, and saccharide composition analysis could be applied to the establishment of herbal medicine marker for identification of different species in various regions.

Discovery of soluble epoxide hydrolase inhibitors from natural products.

With the goal of developing soluble epoxide hydrolase (sEH) inhibitors with novel chemical structures, the sEH inhibitory activities of 30 natural compounds were evaluated using both a fluorescent substrate, 3-phenyl-cyano(6-methoxy-2-naphthalenyl)methyl ester- 2-oxiraneacetic acid, and a physiological substrate, 14,15-epoxyeicosatrienoic acid. To evaluate the selectivity of sEH inhibition, the inhibition of microsomal epoxide hydrolase (mEH), which plays a critical role in detoxification of toxic epoxides, was determined using human liver microsomes. Honokiol and ß-amyrin acetate, isolated from Magnolia officinalis and Acer mandshuricum, respectively, displayed strong inhibition of sEH activity, with respective IC50 values of 0.57 µM and 3.4 µM determined using the fluorescent substrate, and 1.7 µM and 6.1 µM determined using 14,15-epoxyeicosatrienoic acid. mEH activity was decreased to 49% or 61% of control activity by 25 µM honokiol or ß-amyrin acetate, respectively. These results suggest that ß-amyrin acetate and honokiol exhibit sEH inhibitory activity, although their sEH selectivity should be improved.

Clinical characteristics of non-small cell lung cancer patients who experienced acquired resistance during gefitinib treatment.

BACKGROUND: The NSCLC patients who experienced good clinical responses to an EGFR-TKI will inevitably develop acquired resistance. A great deal of research is being carried out to discover the molecular mechanisms underlying this resistance. In comparison, few studies have been conducted to find out about the clinical characteristics of acquired resistance in the patients who had responded to an EGFR-TKI. Herein we investigated clinical characteristics of NSCLC patients who experienced acquired resistance during gefitinib therapy. PATIENTS AND METHODS: We reviewed NSCLC patients who showed a clinical benefit from initial gefitinib therapy. All clinical data were obtained from 11 centers of Korean Molecular Lung Cancer Group (KMLCG). The clinical manifestations of acquired resistance, time to progression (TTP), and post-progression survival (PPS) after gefitinib failure were analyzed retrospectively. RESULTS: A total of 417 patients were recruited. Median TTP was 10.2 months (95% CI, 9.5-10.9). TTP showed a significant longer duration in female, non-smoker, and patients with adenocarcinoma. At the time of acquired resistance, 63.3% of the patients showed symptomatic deterioration. Sites of disease progression were as follows: primary lung lesion in 58.4%, previous metastasis in 38.3%, and new metastasis in 54.2%. Patients with EGFR wild type showed a tendency of higher frequency in symptomatic deterioration and newly development of CNS metastasis compared with patients with EGFR mutation. There was a significant difference in newly development of lung metastasis between patients with exon 19 deletion and those with L858R mutation (41.4% vs. 6.3%, p=0.02). PPS was 8.9 months (95% CI, 7.4-10.4). Smoking history, PS, new CNS lesion and subsequent chemotherapy were independent factors for PPS. CONCLUSION: This study suggests that clinical manifestations of acquired resistance may be different according to EGFR mutation status and EGFR mutation genotype. In addition, subsequent chemotherapy confers clinical benefit in terms of PPS in NSCLC patients who experienced acquired resistance after gefitinib therapy.

Selective and quantitative cell detection based both on aptamers and the conventional cell-staining methods.

Aptamer-based biochips for selective cell detection and quantitation in combination of the recent biochip technology and the conventional cell staining methods are described. Using a model system comprising HER2- or PSMA-positive cells as the analytes and single-stranded RNA aptamers specific for HER2 or PSMA as immobilized ligands on chips, we could demonstrate that aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. In particular, our PSMA-specific sensor was found to have the characteristics of good stability, reproducibility and reusability, with detection limit as low as 10(3) LNCaP cells. In conclusion, we could show the suitability of nucleic acid aptamers as low molecular weight receptors on biochips for sensitive and specific cell detection and quantitation for future diagnostics development.

Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer.

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are recommended as first-line therapy in patients with advanced, recurrent, or metastatic non-squamous non-small cell lung cancer (NSCLC) that have active EGFR mutations. The importance of rapid and sensitive methods for the detection of EGFR mutations is emphasized. The aim of this study is to examine the EGFR mutational status by both direct DNA sequencing and peptide nucleic acid (PNA)-mediated real-time PCR clamping and to evaluate the correlation between the EGFR mutational status and the clinical response to EGFR-tyrosine kinase inhibitors. Clinical specimens from 240 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21. All clinical data and tumor specimens were obtained from 8 centers of the Korean Molecular Lung Cancer Group (KMLCG). After genomic DNA was extracted from paraffin-embedded tissue specimens, we performed PNA-mediated real-time PCR clamping and direct DNA sequencing for the detection of EGFR mutations. Of 240 tumor samples, PNA-mediated PCR clamping was used to detect genomic alterations in 83 (34.6%) samples, including 61 identified by sequencing and 22 additional samples (10 in exon 19, 9 in exon 21, and 3 in both exons); direct DNA sequencing was used to identify a total of 63 (26.3%) mutations that contained 40 deletion mutations in exon 19 (63.5%) and 18 substitution mutations (28.6%) in exon 21. PNA-mediated PCR clamping was used to identify more mutations than clinical direct sequencing, whereas clinical outcomes were not significantly different between the groups harboring activating mutations detected by each method. These data suggest that PNA-mediated real-time PCR clamping exhibits high sensitivity and is a simple procedure relative to direct DNA sequencing that is a useful screening tool for the detection of EGFR mutations in clinical settings.