Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH₄)₂SO₄ were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL⁻¹.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide: A promising positron emission tomography ligand for fatty acid amide hydrolase

To visualize fatty acid amide hydrolase (FAAH) in brain in vivo, we developed a novel positron emission tomography (PET) ligand N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[¹¹C]methylphenyl)thiazol-2-yl]-1-carboxamide ([¹¹C]DFMC, [¹¹C]1). DFMC (1) was shown to have high binding affinity (IC₅₀: 6.1 nM) for FAAH. [¹¹C]1 was synthesized by C–¹¹C coupling reaction of arylboronic ester 2 with [¹¹C]methyl iodide in the presence of Pd catalyst. At the end of synthesis, [¹¹C]1 was obtained with a radiochemical yield of 20 ± 10% (based on [¹¹C]CO₂, decay-corrected, n = 5) and specific activity of 48–166 GBq/μmol. After the injection of [¹¹C]1 in mice, high uptake of radioactivity (>2% ID/g) was distributed in the lung, liver, kidney, and brain, organs with high FAAH expression. PET images of rat brains for [¹¹C]1 revealed high uptakes in the cerebellar nucleus (SUV = 2.4) and frontal cortex (SUV = 2.0), two known brain regions with high FAAH expression. Pretreatment with the FAAH-selective inhibitor URB597 reduced the brain uptake. Higher than 90% of the total radioactivity in the rat brain was irreversible at 30 min after the radioligand injection. The present results indicate that [¹¹C]1 is a promising PET ligand for imaging of FAAH in living brain.     

Effect of essential oils,such as raspberry ketone and its derivatives,on antiandrogenic activity based on in vitro reporter gene assay

The effect of essential oils, such as raspberry ketone, on androgen (AR) receptor was investigated using a MDA-kb2 human breast cancer cell line for predicting potential AR activity. Among them, eugenol had the highest AR antagonistic activity with its IC₅₀ value of 19 μM. Raspberry ketone, which has threefold higher anti-obese activity than that of capsaicin, also had AR antagonist activity with its IC₅₀ value of 252 μM. Based on these findings, a more precise CoMFA model was proposed as follows: pIC₅₀ [log (1/IC₅₀)] = 3.77 + [CoMFA field terms] (n = 39, s = 0.249, r² = 0.834, s_cv = 0.507, q² = 0.311 (three components).     

Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria,erythrocytes and tumor cells

The host-defense peptide, esculentin-2CHa (GFSSIFRGVA¹⁰KFASKGLGK D²⁰LAKLGVDLVA³⁰ CKISKQC) shows potent (MIC ≤ 6 μM) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC₅₀ = 150 μM) and human non-small cell lung adenocarcinoma A549 cells (LC₅₀ = 10 μM). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-α production by peritoneal macrophages. Effects on IL-6 and IL-1β production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys³¹ and Cys³⁷ residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC₅₀ = 11 μM) and A549 cells (LC₅₀ = 3 μM).     

Ligand-based CoMFA and CoMSIA studies on chromone derivatives as radical scavengers

The antioxidant activity for a series of chromone compounds, evaluated by DPPH free radical scavenging assay, were subjected to 3D-QSAR studies using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). All 48 chromone derivatives were geometry optimized by AM1 and HF/6-31G^* calculations. The CoMFA and CoMSIA results were compared between different alignment strategies. The best CoMFA model obtained from HF/6-31G^* optimization with field fit alignment gave cross-validated r² (q²) = 0.821, noncross-validated r² = 0.987, S = 0.095, and F = 388.255. The best CoMSIA model derived from AM1 optimized structures and superimposition alignment gave q² = 0.876, noncross-validated r² = 0.976, S = 0.129, and F = 208.073, including electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. The contour maps provide the fruitful structure–radical scavenging activity relationships which are useful for designing new compounds with higher activity.     

Synthesis and biological activity of aminophthalazines and aminopyridazines as novel inhibitors of PGE2 production in cells

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE₂ in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE₂ reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R¹ = H, R² = p-CH₃O) exhibited the most potent activity in cells (EC₅₀ = 0.02 μM) and minimal inhibition of COX-2 activity (3% at 5 μM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.     

Quantifying human disturbance on antipredator behavior and flush initiation distance in yellow-bellied marmots

Human disturbance may differentially affect the behavior of wild animals and such behavioral perturbations may have fitness consequences. To understand the effects of specific types of human disturbance on antipredator behavior, a behavior whose performance enhances survival, we studied yellow-bellied marmots (Marmota flaviventris). We quantified both antipredator vigilance and the flight initiation distance of the marmots to an approaching human in six different colony sites where we also quantified the frequency and type of human visitation. We developed an analysis framework, using linear mixed models, and found that: (1) when the presence of motorized vehicles and bicycles was high, marmots increased the proportion of time spent vigilant (pseudo R² = 0.33 and 0.31 for motorized vehicles and bicycles, P < 0.05) and decreased the time spent foraging (pseudo R² = 0.29 and 0.23 for motorized vehicles and bicycles, P < 0.05), (2) there was no significant effect of the presence of pedestrians on the time allocated to vigilance and foraging (pseudo R² = 0.25 and 0.19, P > 0.05), (3) marmots decreased the flight initiation distance as disturbance of motorized vehicles (pseudo R² = 0.85) and pedestrians (pseudo R² = 0.84) increased (P < 0.05), and (4) when we considered bicycles as the disturbance, juveniles tolerated closer approaches than adults or yearlings (P < 0.001). Marmots thus responded to some human disturbance by adjusting time spent in foraging and shortening the tolerance distance. Since these behavioral responses could have significant implications for survival and reproduction, we should generally view human disturbance as something that can influence natural antipredator behavior. Importantly, based on an understanding of the differential effects of human activities on wildlife, reducing human disturbance should be taken into account for wildlife management. In addition, our approach will be useful to quantify differential effects of humans on wildlife and to enhance our ability to manage those impacts.     

Recreational physical activity and risk of papillary thyroid cancer among women in the California Teachers Study

Purpose: Little is known about the relationship between physical activity and thyroid cancer risk, and few cohort data on this association exist. Thus, the present study aimed to prospectively examine long-term activity and risk of papillary thyroid cancer among women. Methods: 116,939 women in the California Teachers Study, aged 22–79 years with no history of thyroid cancer at cohort entry, were followed from 1995–1996 through 2009; 275 were diagnosed with invasive papillary thyroid cancer. Cox proportional-hazards regression provided relative risk (RR) estimates and 95% confidence intervals (CI) for associations between thyroid cancer and combined strenuous and moderate recreational physical activity both in the long-term (high school through age 54 years or current age if younger than 54 years) and recently (during the three years prior to joining the cohort). Results: Overall, women whose long-term recreational physical activity averaged at least 5.5 MET-hours/week (i.e. were active) had a non-significant 23% lower risk of papillary thyroid cancer than inactive women (RR = 0.77, 95% CI: 0.57, 1.04). RR estimates were stronger among normal weight or underweight women (body mass index, BMI < 25.0 kg/m², trend p = 0.03) than among overweight or obese women (trend p = 0.35; homogeneity-of-trends p = 0.03). A similar pattern of risk was observed for recent activity (BMI < 25 kg/m², trend p = 0.11; BMI ≥ 25 kg/m², trend p = 0.16; homogeneity-of-trends p = 0.04). Associations for long-term activity did not appear to be driven by activity in any particular life period (e.g. youth, adulthood). Conclusions: Long-term physical activity may reduce papillary thyroid cancer risk among normal weight and underweight women.     

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC₅₀ = 0.5 nM) with a strong binding to sEH (K_i = 3.1 nM) and a moderately long residence time on the enzyme (k_off = 1.05 × 10⁻³ s⁻¹; t_1/2 = 11 min).     

Syntheses and pharmacological characterization of novel thiazole derivatives as potential mGluR5 PET ligands

Four novel thiazole containing ABP688 derivatives were synthesized and evaluated for their binding affinity towards the metabotropic glutamate receptor subtype 5 (mGluR5). (E)-3-((2-(Fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime (FTECMO), the ligand with the highest binding affinity (K_i = 5.5 ± 1.1 nM), was labeled with fluorine-18. [¹⁸F]-FTECMO displayed optimal lipophilicity (log D_pH7.4 = 1.6 ± 0.2) and high stability in rat and human plasma as well as sufficient stability in rat liver microsomes. In vitro autoradiography with [¹⁸F]-FTECMO revealed a heterogeneous and displaceable binding in mGluR5-rich brain regions. PET imaging with [¹⁸F]-FTECMO in Wistar rats, however, showed low brain uptake. Uptake of radioactivity into the skull was observed suggesting in vivo defluorination. Thus, although [¹⁸F]-FTECMO is an excellent ligand for the detection of mGluR5 in vitro, its in vivo characteristics are not optimal for the imaging of mGluR5 in rats in vivo.     

Antimycobacterial activity evaluation,time-kill kinetic and 3D-QSAR study of C-(3-aminomethyl-cyclohexyl)-methylamine derivatives

A series of C-(3-aminomethyl-cyclohexyl)-methylamine derivatives were synthesized and evaluated for their antitubercular activity. Some of the compounds exhibited potent activity against Mycobacterium tuberculosis H37Rv. One of the compound having t-butyl at para position of the benzene ring showed excellent activity even better than the standard drug ethambutol with MIC value 1.1 ± 0.2 μM. The time-kill kinetics study of two most active compounds showed rapid killing of the M. tuberculosis within 4 days. Additionally atom-based quantitative structure–activity relationship (QSAR) model was developed that gave a statistically satisfying result (R²) = 0.92, Q² = 0.75, Pearson-R = 0.96 and effectively predicts the anti-tuberculosis activity of training and test set compounds.     

A multiple free-radical scavenging (MULTIS) study on the antioxidant capacity of a neuroprotective drug,edaravone as compared with uric acid,glutathione, and trolox

Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) is a neuroprotective drug that has been used for brain ischemia injury treatment. Because its activity is speculated to be due to free radical scavenging activity, we carried out a quantitative determination of edaravone’s free radical scavenging activity against multiple free radical species. Electron spin resonance (ESR) spin trapping-based multiple free-radical scavenging (MULTIS) method was employed, where target free radicals were hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen. Edaravone showed relatively high scavenging abilities against hydroxyl radical (scavenging rate constant k = 2.98 × 10¹¹ M⁻¹ s⁻¹), singlet oxygen (k = 2.75 × 10⁷ M⁻¹ s⁻¹), and methyl radical (k = 3.00 × 10⁷ M⁻¹ s⁻¹). Overall, edaravone’s scavenging activity against multiple free radical species is as robust as other known potent antioxidant such as uric acid, glutathione, and trolox. A radar chart illustration of the MULTIS activity relative to uric acid, glutathione, and trolox indicates that edaravone has a high and balanced antioxidant activity with low specificity.     

Electromyographic and mechanomyographic responses across repeated maximal isometric and concentric muscle actions of the leg extensors

The purpose of the present study was to examine the patterns of responses for torque, electromyographic (EMG) amplitude, EMG mean power frequency (MPF), mechanomyographic (MMG) amplitude, and MMG MPF across 30 repeated maximal isometric (ISO) and concentric (CON) muscle actions of the leg extensors. Twelve female subjects (21.1 ± 1.4 yrs; 63.3 ± 7.4 kg) performed ISO and CON fatigue protocols with EMG and MMG signals recorded from the vastus lateralis. The relationships for torque, EMG amplitude, EMG MPF, MMG amplitude, and MMG MPF versus repetition number were examined using polynomial regression. The results indicated there were decreases (p < 0.05) across the ISO muscle actions for torque (r² = 0.95), EMG amplitude (R² = 0.44), EMG MPF (r² = 0.62), and MMG MPF (r² = 0.48), but no change in MMG amplitude (r² = 0.07). In addition, there were decreases across the CON muscle actions for torque (R² = 0.97), EMG amplitude (R² = 0.46), EMG MPF (R² = 0.86), MMG amplitude (R² = 0.44), and MMG MPF (R² = 0.80). Thus, the current findings suggested that the mechanisms of fatigue and motor control strategies used to modulate torque production were similar between maximal ISO and CON muscle actions.     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

Whole crop rice silage: Predictions of yield and content of metabolizable energy,metabolizable protein and other nutrients for dairy cows from crop maturity and botanical fractions at harvest

The study investigated the suitability of stage of maturity and botanical fractions of whole crop rice (WCR) to predict yield and nutritive value of ensiled WCR for dairy cows. Eight varieties of WCR (i.e., Akichikara, Fukuhibiki, Habataki, Hamasari, Hokuriku 168, Kusanami, Tamakei 96, Yumetoiro) were harvested at four stages of maturity (i.e., 10, 22, 34, 45 days after flowering [DAF]) and ensiled. Dry matter (DM) yield at each harvest was determined. Silage samples were fractionated into four botanical fractions being: leaf blade, leaf sheath, stem and head. Silage samples were also analyzed for chemical composition, fermentation characteristics, in situ DM and N disappearance. Metabolizable energy (ME) and metabolizable protein (MP) content of samples were estimated according to Terada et al. (1988) and AFRC (1993), respectively. Relationships between maturity or proportions of botanical fractions and contents of WCR silage in terms of DM, ME and MP, and their yields, were estimated by correlation and regression analysis. Stage of maturity was positively related (P<0.001) to ME content (R² = 0.46; y = 4.53 + 0.08X) and MP content (R² = 0.56; y = 22.26 + 0.76X), and DM yield (R² = 0.63; y = 9.21 + 0.12X), ME yield (R² = 0.68, y = 36931 + 1708X) and MP yield (R² = 0.72, y = 161.0 + 14.15X) of WCR. Proportion of leaf was negatively related to yields and nutritive value of ensiled WCR, whilst proportion of head was positively related (P<0.05 to <0.001). Proportion of head was best related to the ME content (R² = 0.72; y = 3.26 + 0.009X), MP content (R² = 0.72; y = 12.31 + 0.079X), and DM yield (R² = 0.41; y = 9.02 + 0.009X), ME yield (R² = 0.76, y = 19494 + 165.5X), and MP yield (R² = 0.75, y = 34.37 + 1.32X) of WCR. Results suggest that to optimize yield and nutritive value, WCR should be ensiled within 40 DAF and the proportion of head should be equal to or more than 500 g per kg DM of WCR silage. Stage of maturity and proportion of head of WCR predict yields of DM, ME and MP of WCR, and their contents, in WCR silage with acceptable accuracy. However, these relationships need to be validated using large data sets and in vivo studies.     

Design,synthesis and biological evaluation of new 2,3-diarylquinoline derivatives as selective cyclooxygenase-2 inhibitors

A new group of 2,3-diarylquinoline derivatives possessing a methylsulfonyl COX-2 pharmacophore at the para-position of the C-2 phenyl ring were synthesized and evaluated as selective COX-2 inhibitors. In vitro COX-1/COX-2 structure–activity relationships were determined by varying the substituents on the C-4 quinoline ring. Among the 2,3-diarylquinolines, 2-(4-(methylsulfonyl) phenyl)-3-phenylquinoline-4-carboxylic acid (8) exhibited the highest potency and selectivity for COX-2 inhibitory activity (COX-2 IC₅₀ = 0.07 μM; selectivity index = 687.1) that was more selective than the reference drug celecoxib (COX-2 IC₅₀ = 0.06 μM; selectivity index = 405). A molecular modeling study where 8 was docked in the binding site of COX-2 indicated that the p-MeSO₂ COX-2 pharmacophore group on the C-2 phenyl ring is oriented in the vicinity of the COX-2 secondary pocket (Arg⁵¹³, Phe⁵¹⁸ and Val⁵²³) and the carboxylic acid substituent can interact with Ser⁵³⁰. The structure activity data acquired indicate that the size and nature of the C-4 quinoline substituent are important for COX-2 inhibitory activity.     

Neuromuscular adaptations predict functional disability independently of clinical pain and psychological factors in patients with chronic non-specific low back pain

Patients with chronic low back pain exhibit characteristics such as clinical pain, psychological symptoms and neuromuscular adaptations. The purpose of this study was to determine the independent contribution of clinical pain, psychological factors and neuromuscular adaptations to disability in patients with chronic low back pain. Clinical pain intensity, pain catastrophizing, fear-avoidance beliefs, anxiety, neuromuscular adaptations to chronic pain and neuromuscular responses to experimental pain were assessed in 52 patients with chronic low back pain. Lumbar muscle electromyographic activity was assessed during a flexion–extension task (flexion relaxation phenomenon) to assess both chronic neuromuscular adaptations and neuromuscular responses to experimental pain during the task. Multiple regressions showed that independent predictors of disability included neuromuscular adaptations to chronic pain (β = 0.25, p = 0.006, sr² = 0.06), neuromuscular responses to experimental pain (β = −0.24, p = 0.011, sr² = 0.05), clinical pain intensity (β = 0.28, p = 0.002, sr² = 0.08) and psychological factors (β = 0.58, p < 0.001, sr² = 0.32). Together, these predictors accounted for 65% of variance in disability (R² = 0.65 p < 0.001). The current investigation revealed that neuromuscular adaptations are independent from clinical pain intensity and psychological factors, and contribute to inter-individual differences in patients’ disability. This suggests that disability, in chronic low back pain patients, is determined by a combination of factors, including clinical pain, psychological factors and neuromuscular adaptations.     

Synthesis and evaluation of copper-64 labeled benzofuran derivatives targeting β-amyloid aggregates

In vivo imaging of β-amyloid (Aβ) aggregates consisting of Aβ(1–40) and Aβ(1–42) peptides by positron emission tomography (PET) contributes to the diagnosis and therapy for Alzheimer’s disease (AD). Because ⁶⁴Cu (t_1/2 = 12.7 h) is a radionuclide for PET with a longer physical half-life than ¹¹C (t_1/2 = 20 min) and ¹⁸F (t_1/2 = 110 min), it is an attractive radionuclide for the development of Aβ imaging probes that are suitable for routine use. In the present study, we designed and synthesized two novel ⁶⁴Cu labeled benzofuran derivatives and evaluated their utility as PET imaging probes for Aβ aggregates. In an in vitro binding assay, 6 and 8 showed binding affinity for Aβ(1–42) aggregates with a K_i value of 33 and 243 nM, respectively. In addition, these probes bound to Aβ plaques deposited in the brain of an AD model mouse in vitro. In a biodistribution experiment using normal mice, these probes showed low brain uptake (0.33% and 0.36% ID/g) at 2 min post-injection. Although refinement to enhance brain uptake is needed, [⁶⁴Cu]6 and [⁶⁴Cu]8 demonstrated the feasibility of developing novel PET probes for imaging Aβ aggregates.