Human Polymerase δ-Interacting Protein 2 (PolDIP2) Inhibits the Formation of Human Tau Oligomers and Fibrils

A central characteristic of Alzheimer’s disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.

Use of immunoglobulin-binding bacterial proteins in immunodetection

One of the aim of this study was to make universal chimeric conjugates to react with both avian and mammalian immunoglobulins in enzyme-linked immunosorbent assays (ELISAs). The periodate method was used in the conjugation process of cross-linking horseradish peroxidase to immunoglobulin-binding proteins (IBP) including staphylococcal protein A (SpA), streptococcal protein G (SpG) and peptostreptococcal protein L (SpL). By mixing up these three conjugates another four hybrid protein conjugates were created including protein LA (SpLA), protein LG (SpLG), protein AG (SpAG) and protein LAG (PLAG). Thirty-five ELISAs were standardized by a probabilistic combination of these immunoreagents. By using a panel of mainly mammalian immunoglobulins their reproducibility was checked by the determination of coefficient of variations (CV) for each one of the IgG-IBP binding. The source of immunoglobulins was their purification by affinity chromatography using a commercially available kit (PURE-1A). The other aim was to immunize chicken with the peptide fragment 254-274 of gp120 to produce anti-HIV peptide hyper-immune egg. Cats and rats were fed these eggs for a determined period until they produced the anti-HIV peptide antibody, which was tested by an indirect SpLA-ELISA and dot blot analysis that corroborated the production of anti-HIV antibodies by the mammalian species including positive humans samples for HIV. We conclude that the single and hybrid immunoglobulin-binding protein were effective in their binding capacity to immunoglobulins from a variety of mammalian species. The potential use of this proteins is in the arena of immunodiagnosis and immunoglobulin detection. Dot blot analysis proves effective in the detection of HIV anti-gp120 antibodies in several animal species. These antibodies can be used as reagents in the development of immunodiagnostic tests or experimental vaccines.

Suggestive Serological Evidence of Infection with Shrew-Borne Imjin Virus (Hantaviridae) in Humans

The pathogenicity of the shrew-borne Imjin virus (MJNV) is unknown. The objective of our study was to find serological evidence of MJNV infection in humans. Partial MJNV nucleocapsid protein (NP) was cloned and expressed as an antigen for double-antigen sandwich ELISA, IgM capture ELISA, and dot blot to detect MJNV specific antibodies in hemorrhagic fever with renal syndrome (HFRS) patients’ and healthy persons’ sera from endemic areas in China. The purified recombinant NP reacted with neither the 90 healthy individuals’ sera from non-endemic areas of MJNV nor the 100 antisera to HFRS-causing virus, indicating that the MJNV NP had no cross-reaction with normal human sera and HFRS-causing viral antibodies. As determined by screening ELISA and dot blot analysis, IgG antibodies against MJNV NP were detected in sera from two of 385 healthy individuals from MJNV-endemic areas, suggesting infection with MJNV or MJNV-like thottimvirus. Based on the suggestive evidence, healthcare workers should be alert to febrile diseases occurring among individuals with exposure to shrew-infested habitats.

Transient Focal Ischemia Significantly Alters the m 6 A Epitranscriptomic Tagging of RNAs in the Brain

Background and Purpose— Adenosine in many types of RNAs can be converted to m 6 A (N 6 -methyladenosine) which is a highly dynamic epitranscriptomic modification that regulates RNA metabolism and function. Of all organs, the brain shows the highest abundance of m 6 A methylation of RNAs. As recent studies showed that m 6 A modification promotes cell survival after adverse conditions, we currently evaluated the effect of stroke on cerebral m 6 A methylation in mRNAs and lncRNAs. Methods— Adult C57BL/6J mice were subjected to transient middle cerebral artery occlusion. In the peri-infarct cortex, m 6 A levels were measured by dot blot analysis, and transcriptome-wide m 6 A changes were profiled using immunoprecipitated methylated RNAs with microarrays (44 122 mRNAs and 12 496 lncRNAs). Gene ontology analysis was conducted to understand the functional implications of m 6 A changes after stroke. Expression of m 6 A writers, readers, and erasers was also estimated in the ischemic brain. Results— Global m 6 A levels increased significantly at 12 hours and 24 hours of reperfusion compared with sham. While 139 transcripts (122 mRNAs and 17 lncRNAs) were hypermethylated, 8 transcripts (5 mRNAs and 3 lncRNAs) were hypomethylated (>5-fold compared with sham) in the ischemic brain at 12 hours reperfusion. Inflammation, apoptosis, and transcriptional regulation are the major biological processes modulated by the poststroke differentially m 6 A methylated mRNAs. The m 6 A writers were unaltered, but the m 6 A eraser (fat mass and obesity-associated protein) decreased significantly after stroke compared with sham. Conclusions— This is the first study to show that stroke alters the cerebral m 6 A epitranscriptome, which might have functional implications in poststroke pathophysiology. Visual Overview— An online visual overview is available for this article.

Discrimination of urinary exosomes from microvesicles by lipidomics using thin layer liquid chromatography (TLC) coupled with MALDI-TOF mass spectrometry

Urinary extracellular vesicles (EVs), including microvesicles and exosomes, play several important roles in cell biology and serve as potential biomarkers in various kidney diseases. Although they have differential biophysical properties, specific biomarkers are required to discriminate these EVs during isolation/purification. The present study aimed to define differential lipidome profiles of urinary microvesicles vs. exosomes. Urine samples collected from eight healthy individuals were pooled and underwent lipid extraction using 2:1(v/v) chloroform/methanol. The recovered lipids were resolved by thin layer liquid chromatography (TLC) and analyzed by MALDI-TOF MS. From three and five TLC bands observed in microvesicles and exosomes, respectively, several fatty acids, glycerolipids and phospholipids were identified from both EVs without clear differential patterns. However, their sphingolipid profiles were unique. Ceramide phosphates (CerP), hexosyl sphingoid bases (HexSph), lactosyl ceramides (LacCer), mannosyl di-PI-ceramides (M(IP)2 C), sulfatides hexosyl ceramide (SHexCer) and sulfatides hexoxyl sphingoid bases (SHexSph) were detectable only in urinary exosomes, whereas phosphatidylinositol ceramides (PI-Cer) were detectable only in urinary microvesicles. The presence of CerP only in urinary exosomes was successfully validated by dot blot analysis. Our extensive lipidome analyses of urinary microvesicles vs. exosomes provide potential lipidome markers to discriminate exosomes from microvesicles and may lead to better understanding of EVs biogenesis.

Serum Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) Level as a Potential Biomarker of Cholangiocarcinoma

Diagnostic and/or prognostic biomarkers for cholangiocarcinoma (CCA) are still insufficient with poor prognosis of patients. To discover a new CCA biomarker, we constructed our secretome database of three CCA cell lines and one control cholangiocyte cell line using GeLC-MS/MS. We selected candidate proteins by five bioinformatics tools for secretome analysis. The inclusion criteria were as follows: having predicted signal peptide or being predicted as non-classically secreted protein; together with having no transmembrane helix and being previously detected in plasma and having the highest number of signal peptide cleavage sites. Eventually, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) was selected for further analysis. To validate APEX1 as a bio-marker for CCA, serum APEX1 levels of 80, 39, and 40 samples collected from CCA, benign biliary diseases (BBD), and healthy control groups, respectively, were measured using dot blot analysis. The results showed that serum APEX1 level in CCA group was significantly higher than that in BBD or healthy control group. Among CCA patients, serum APEX1 level was significantly higher in patients having metastasis than in those without metastasis. The higher level of serum APEX1 was correlated with the shorter survival time of the patients. Serum APEX1 level might be a diagnostic and prognostic biomarker for CCA.

An acetylation mimicking mutation, K274Q, in tau imparts neurotoxicity by enhancing tau aggregation and inhibiting tubulin polymerization

In Alzheimer's disease, tau is predominantly acetylated at K174, K274, K280, and K281 residues. The acetylation of K274-tau is linked with memory loss and dementia. In this study, we have examined the molecular mechanism of the toxicity of acetylated K274-tau. We incorporated an acetylation mimicking mutation at K274 (K→Q) residue of tau. The mutation (K274Q) strongly reduced the ability of tau to bind to tubulin and also to polymerize tubulin while K274R mutation did not reduce the ability of tau either to bind or polymerize tubulin. In addition, K274Q-tau displayed a higher aggregation propensity than wild-type tau as evident from thioflavin S fluorescence, tryptophan fluorescence, and electron microscopic images. Furthermore, dynamic light scattering, atomic force microscopy, and dot blot analysis using an oligomer-specific antibody suggested that K274Q mutation enhanced the oligomerization of tau. The K274Q mutation also strongly decreased the critical concentration for the liquid–liquid phase separation of tau. The oligomeric forms of K274Q-tau were found to be more toxic than wild tau to neuroblastoma cells. Using circular dichroism and fluorescence spectroscopy, we provide evidence indicating that the acetylation mimicking mutation (K274Q) induced conformational changes in tau. The results suggested that the acetylation of tau at 274 residues can increase tau aggregation and enhance the cytotoxicity of tau oligomers.