Establishing In-House Cutoffs of CSF Alzheimer’s Disease Biomarkers for the AT(N) Stratification of the Alzheimer Center Barcelona Cohort

Background: Clinical diagnosis of Alzheimer’s disease (AD) increasingly incorporates CSF biomarkers. However, due to the intrinsic variability of the immunodetection techniques used to measure these biomarkers, establishing in-house cutoffs defining the positivity/negativity of CSF biomarkers is recommended. However, the cutoffs currently published are usually reported by using cross-sectional datasets, not providing evidence about its intrinsic prognostic value when applied to real-world memory clinic cases. Methods: We quantified CSF Aβ1-42, Aβ1-40, t-Tau, and p181Tau with standard INNOTEST^® ELISA and Lumipulse G^® chemiluminescence enzyme immunoassay (CLEIA) performed on the automated Lumipulse G600II. Determination of cutoffs included patients clinically diagnosed with probable Alzheimer’s disease (AD, n = 37) and subjective cognitive decline subjects (SCD, n = 45), cognitively stable for 3 years and with no evidence of brain amyloidosis in 18F-Florbetaben-labeled positron emission tomography (FBB-PET). To compare both methods, a subset of samples for Aβ1-42 (n = 519), t-Tau (n = 399), p181Tau (n = 77), and Aβ1-40 (n = 44) was analyzed. Kappa agreement of single biomarkers and Aβ1-42/Aβ1-40 was evaluated in an independent group of mild cognitive impairment (MCI) and dementia patients (n = 68). Next, established cutoffs were applied to a large real-world cohort of MCI subjects with follow-up data available (n = 647). Results: Cutoff values of Aβ1-42 and t-Tau were higher for CLEIA than for ELISA and similar for p181Tau. Spearman coefficients ranged between 0.81 for Aβ1-40 and 0.96 for p181TAU. Passing–Bablok analysis showed a systematic and proportional difference for all biomarkers but only systematic for Aβ1-40. Bland–Altman analysis showed an average difference between methods in favor of CLEIA. Kappa agreement for single biomarkers was good but lower for the Aβ1-42/Aβ1-40 ratio. Using the calculated cutoffs, we were able to stratify MCI subjects into four AT(N) categories. Kaplan–Meier analyses of AT(N) categories demonstrated gradual and differential dementia conversion rates (p = 9.815⁻²⁷). Multivariate Cox proportional hazard models corroborated these findings, demonstrating that the proposed AT(N) classifier has prognostic value. AT(N) categories are only modestly influenced by other known factors associated with disease progression. Conclusions: We established CLEIA and ELISA internal cutoffs to discriminate AD patients from amyloid-negative SCD individuals. The results obtained by both methods are not interchangeable but show good agreement. CLEIA is a good and faster alternative to manual ELISA for providing AT(N) classification of our patients. AT(N) categories have an impact on disease progression. AT(N) classifiers increase the certainty of the MCI prognosis, which can be instrumental in managing real-world MCI subjects.     

Application of Fast Gas Chromatography–Mass Spectrometry in Combination with the QuEChERS Method for the Determination of Pesticide Residues in Fruits and Vegetables

A fast gas chromatography–mass spectrometry method has been developed for multiresidue determination of up to 56 pesticides in fruits and vegetables in a chromatographic run time of <10 min, using a single quadrupole mass spectrometer operating in selected ion monitoring mode. The well-known acetate-buffering version of the QuEChERS method has been used for sample preparation. Programmable temperature vaporizer injection of 3 μL allowed reaching limits of detection between 0.15 and 15 μg/kg for most compounds in the sample matrices tested. The applicability of the method has been evaluated in apple, orange, carrot, and tomato. Recoveries at three fortification levels (0.01, 0.1 and 0.5 mg/kg) ranged from 70 to 120 % for most compounds, with relative standard deviations below 20 % in all cases. The developed method has been applied to fruit and vegetable samples from different Spanish provinces.     

Determination of polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng

The total polyphenol content and the antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng were determined by Folin–Ciocalteu reagent, and DPPH, FRAP, and TEAC methods, respectively. The crude extract (CE) of kudingcha and its four fractions of chloroform (CfF), ethyl acetate (EaF), n-butanol (nBF), and water (WtF) were prepared and subjected to antioxidant evaluation and analysis by high performance liquid chromatography. The extracts of kudingcha contained large amounts of caffeoylquinic acid (CQA) derivatives, including 3-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA, and showed potent antioxidant activity. The antioxidant activities of CE and its fractions decreased in the order of EaF > nBF > CE > WtF > CfF, according to the DPPH assay and FRAP assay, which were the same, with the exception of the rank order of CfF and WtF, as the TEAC assay. Furthermore, a satisfactory correlation between total polyphenol content and antioxidant activity was observed.     

The anticarcinogenic potential of essential oil and aqueous infusion from caper (Capparis spinosa L.)

The present study assessed the influence of essential oil and aqueous infusion from wild-grown caper (Capparis spinosa L.) on cell growth, NF-κB activation, apoptosis and cell cycle in the human colon carcinoma cell line, HT-29. Methyl isothiocyanate (92.06%), a degradation product of glucosinolate glucocapparin, was detected as major component of essential oil from caper leaves and flower buds. Aqueous infusion of caper showed an interesting and variegate compositional pattern containing several phenolic compounds, among which a flavonol glycoside, rutin (quercetin 3-O-rutinoside, 50.7%) and 5-caffeoyl-quinic acid (chlorogenic acid, 17.5%) were detected as dominant. Caper essential oil and aqueous infusion showed time- and dose-dependent high inhibitory effect on HT-29 cell proliferation. In addition, they induced the inhibition on nuclear factor κB (NF-κB) activity in a dose-dependent manner, while they did not show any effect on apoptosis in HT-29 cells. Flow cytometric analysis indicated that treatment with caper essential oil and aqueous infusion resulted in G₂/M cell cycle arrest in a dose-dependent manner. Presented results suggest that caper contains volatile and non-volatile compounds which potentially can play an important role in colon cancer prevention.     

Intracellular silver accumulation in Chlamydomonas reinhardtii upon exposure to carbonate coated silver nanoparticles and silver nitrate

The intracellular silver accumulation ({Ag}(in)) upon exposure to carbonate coated silver nanoparticles (AgNP, 0.5-10 μM, average diameter 29 nm) and silver nitrate (20-500 nM) was examined in the wild type and in the cell wall free mutant of the green alga Chlamydomonas reinhardtii at pH 7.5. The {Ag}(in) was measured over time up to 1 h after a wash procedure to remove silver ions (Ag(+)) and AgNP from the algal cell surface. The {Ag}(in) increased with increasing exposure time and with increasing AgNP and AgNO(3) concentrations in the exposure media, reaching steady-state concentrations between 10(-5) and 10(-3) mol L(cell)(-1). According to estimated kinetic parameters, high Ag(+) bioconcentration factors were calculated (>10(3) L L(cell)(-1)). Higher accumulation rate constants were assessed in the cell wall free mutant, indicating a protective role of the cell wall in limiting Ag(+) uptake. The bioavailability of AgNP was calculated to be low in both strains relative to Ag(+), suggesting that AgNP internalization across the cell membrane was limited.     

Engineered Neutral Phosphorous Dendrimers Protect Mouse Cortical Neurons and Brain Organoids from Excitotoxic Death

Nanoparticles are playing an increasing role in biomedical applications. Excitotoxicity plays a significant role in the pathophysiology of neurodegenerative diseases, such as Alzheimer’s or Parkinson’s disease. Glutamate ionotropic receptors, mainly those activated by N-methyl-D-aspartate (NMDA), play a key role in excitotoxic death by increasing intraneuronal calcium levels; triggering mitochondrial potential collapse; increasing free radicals; activating caspases 3, 9, and 12; and inducing endoplasmic reticulum stress. Neutral phosphorous dendrimers, acting intracellularly, have neuroprotective actions by interfering with NMDA-mediated excitotoxic mechanisms in rat cortical neurons. In addition, phosphorous dendrimers can access neurons inside human brain organoids, complex tridimensional structures that replicate a significant number of properties of the human brain, to interfere with NMDA-induced mechanisms of neuronal death. Phosphorous dendrimers are one of the few nanoparticles able to gain access to the inside of neurons, both in primary cultures and in brain organoids, and to exert pharmacological actions by themselves.     

Design and Development of a Bimodal Optical Instrument for Simultaneous Vibrational Spectroscopy Measurements

Vibrational spectroscopy techniques are widely used in analytical chemistry, physics and biology. The most prominent techniques are Raman and Fourier-transform infrared spectroscopy (FTIR). Combining both techniques delivers complementary information of the test sample. We present the design, construction, and calibration of a novel bimodal spectroscopy system featuring both Raman and infrared measurements simultaneously on the same sample without mutual interference. The optomechanical design provides a modular flexible system for solid and liquid samples and different configurations for Raman. As a novel feature, the Raman module can be operated off-axis for optical sectioning. The calibrated system demonstrates high sensitivity, precision, and resolution for simultaneous operation of both techniques and shows excellent calibration curves with coefficients of determination greater than 0.96. We demonstrate the ability to simultaneously measure Raman and infrared spectra of complex biological material using bovine serum albumin. The performance competes with commercial systems; moreover, it presents the additional advantage of simultaneously operating Raman and infrared techniques. To the best of our knowledge, it is the first demonstration of a combined Raman-infrared system that can analyze the same sample volume and obtain optically sectioned Raman signals. Additionally, quantitative comparison of confocality of backscattering micro-Raman and off-axis Raman was performed for the first time.