Disturbed Ca²⁺ homeostasis increases QC expression and enzyme activity.

<p>A. Differentiated SK-N-SH cells were treated with different concentrations TG as indicated. Shown are the average + SD of normalized QC mRNA levels of n = 6 from two independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. B. Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h. The enzymatic activity of QC activity was determined in protein lysates as described in materials and methods, activity in untreated cells is set to 1. Shown is average +SEM of n = 15 from 5 independent experiments. Asterisks indicate a significant difference compared to control (*p≤0.001, **p≤0.0001).</p

QC expression is not upregulated by Aβ or the UPR.

<p>A. Differentiated SK-N-SH cells were treated with 1 µM and 2 µM oligomeric (O) and fibrillary (F) Aβ_1–42 for 24 h. Shown is the average + SD of normalized QC mRNA levels of triplicates from a representative experiment B. Differentiated SK-N-SH cells were treated with different UPR inducers, TM (0.2 µg/µl and 0.5 µg/µl), TG (1 µM and 2 µM) and DD (20 mM) for 16 h. Shown is the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. Normalized BiP mRNA levels are shown as positive control for UPR induction (n = 6 from two independent experiments). The expression levels were normalized to eEF2α mRNA, the levels in untreated cells are set to 1. Asterisks indicate a significant difference compared with control (*p≤0.01, **p≤0.001, ***p≤0.0001).</p

Post-mortem brain material used in this study.

<p>Listed are clinical diagnosis (CON = control, AD = Alzheimer’s disease), Braak stage, gender, age (in years), ApoE genotype, post-mortem interval (PMI, in hours: minutes).</p

Primers and probes used for qPCR 480 light cycler.

<p>Probe numbers refer to numbers in the Roche universal probe library.</p

ER Ca²⁺ depletion increases QC levels.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p≤0.0001).</p

QC mRNA expression is increased in earliest stages of AD pathology.

<p>Expression of QC mRNA was determined in a cohort of patients with varying stages of AD pathology (see materials and methods for details). CON and AD refer to the clinical diagnosis, BS is Braak score for tau pathology, PL is the plaqueload in the hippocampus/entorhinal cortex. Shown is a box-plot of results of the pathological groups as indicated in hippocampus/entorhinal cortex. The expression levels were normalized to eEF2α mRNA. Kruskall-Wallis test was used to evaluate differences between groups followed by the Mann-Whitney U test, to test differences between pairs of groups.</p

Induction of c-fos and c-jun precedes increased QC expression.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for the times indicated. Shown is the average + SD of normalized QC (A), c-fos (B) and c-jun (C) mRNA levels of triplicates from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05; **p≤0.001; ***p≤0.0001).</p

Induction of c-fos by ER Ca²⁺ depletion precedes QC expression.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for 5 h and 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of triplicates of normalized QC (A) and c-fos (B) mRNA levels from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05, **p≤0.02 ***p≤0.001).</p

PEGylated Nanoparticles Bind to and Alter Amyloid-Beta Peptide Conformation: Toward Engineering of Functional Nanomedicines for Alzheimer’s Disease

We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aβ_1–42) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the interaction with monomeric and soluble oligomeric forms of Aβ_1–42 peptide was demonstrated by capillary electrophoresis, surface plasmon resonance, thioflavin T assay, and confocal microscopy, where the binding affected peptide aggregation kinetics. The capture of peptide by NPs in serum was also evidenced by fluorescence spectroscopy and ELISA. Moreover, <i>in silico</i> and modeling experiments highlighted the mode of PEG interaction with the Aβ_1–42 peptide and its conformational changes at the nanoparticle surface. Finally, Aβ_1–42 peptide binding to NPs affected neither complement activation in serum nor apolipoprotein-E (Apo-E) adsorption from the serum. These observations have crucial implications in NP safety and clearance kinetics from the blood. Apo-E deposition is of prime importance since it can also interact with the Aβ_1–42 peptide and increase the affinity of NPs for the peptide in the blood. Collectively, our results suggest that these engineered long-circulating NPs may have the ability to capture the toxic forms of the Aβ_1–42 peptide from the systemic circulation and potentially improve Alzheimer’s disease condition through the proposed “<i>sink effect</i>”

Highly Sensitive Single Domain Antibody–Quantum Dot Conjugates for Detection of HER2 Biomarker in Lung and Breast Cancer Cells

Despite the widespread availability of immunohistochemical and other methodologies for screening and early detection of lung and breast cancer biomarkers, diagnosis of the early stage of cancers can be difficult and prone to error. The identification and validation of early biomarkers specific to lung and breast cancers, which would permit the development of more sensitive methods for detection of early disease onset, is urgently needed. In this paper, ultra-small and bright nanoprobes based on quantum dots (QDs) conjugated to single domain anti-HER2 (human epidermal growth factor receptor 2) antibodies (sdAbs) were applied for immunolabeling of breast and lung cancer cell lines, and their performance was compared to that of anti-HER2 monoclonal antibodies conjugated to conventional organic dyes Alexa Fluor 488 and Alexa Fluor 568. The sdAbs–QD conjugates achieved superior staining in a panel of lung cancer cell lines with differential HER2 expression. This shows their outstanding potential for the development of more sensitive assays for early detection of cancer biomarkers