A blood-based signature of cerebrospinal fluid Aß1-42 status.

It is increasingly recognized that Alzheimer's disease (AD) exists before dementia is present and that shifts in amyloid beta occur long before clinical symptoms can be detected. Early detection of these molecular changes is a key aspect for the success of interventions aimed at slowing down rates of cognitive decline. Recent evidence indicates that of the two established methods for measuring amyloid, a decrease in cerebrospinal fluid (CSF) amyloid ß1-42 (Aß1-42) may be an earlier indicator of Alzheimer's disease risk than measures of amyloid obtained from Positron Emission Tomography (PET). However, CSF collection is highly invasive and expensive. In contrast, blood collection is routinely performed, minimally invasive and cheap. In this work, we develop a blood-based signature that can provide a cheap and minimally invasive estimation of an individual's CSF amyloid status using a machine learning approach. We show that a Random Forest model derived from plasma analytes can accurately predict subjects as having abnormal (low) CSF Aß1-42 levels indicative of AD risk (0.84 AUC, 0.78 sensitivity, and 0.73 specificity). Refinement of the modeling indicates that only APOEε4 carrier status and four plasma analytes (CGA, Aß1-42, Eotaxin 3, APOE) are required to achieve a high level of accuracy. Furthermore, we show across an independent validation cohort that individuals with predicted abnormal CSF Aß1-42 levels transitioned to an AD diagnosis over 120 months significantly faster than those with predicted normal CSF Aß1-42 levels and that the resulting model also validates reasonably across PET Aß1-42 status (0.78 AUC). This is the first study to show that a machine learning approach, using plasma protein levels, age and APOEε4 carrier status, is able to predict CSF Aß1-42 status, the earliest risk indicator for AD, with high accuracy.

Stage-based Variation in the Effect of Primary Tumor Side on All Stages of Colorectal Cancer Recurrence and Survival.

BACKGROUND: Multiple studies have defined the prognostic and potential predictive significance of the primary tumor side in metastatic colorectal cancer (CRC). However, the currently available data for early-stage disease are limited and inconsistent. MATERIALS AND METHODS: We explored the clinicopathologic, treatment, and outcome data from a multisite Australian CRC registry from 2003 to 2016. Tumors at and distal to the splenic flexure were considered a left primary (LP). RESULTS: For the 6547 patients identified, the median age at diagnosis was 69 years, 55% were men, and most (63%) had a LP. Comparing the outcomes for right primary (RP) versus LP, time-to-recurrence was similar for stage I and III disease, but longer for those with a stage II RP (hazard ratio [HR], 0.68; 95% confidence interval [CI], 0.52-0.90; P < .01). Adjuvant chemotherapy provided a consistent benefit in stage III disease, regardless of the tumor side. Overall survival (OS) was similar for those with stage I and II disease between LP and RP patients; however, those with stage III RP disease had poorer OS (HR, 1.30; 95% CI, 1.04-1.62; P < .05) and cancer-specific survival (HR, 1.55; 95% CI, 1.19-2.03; P < .01). Patients with stage IV RP, whether de novo metastatic (HR, 1.15; 95% CI, 0.95-1.39) or relapsed post-early-stage disease (HR, 1.35; 95% CI, 1.11-1.65; P < .01), had poorer OS. CONCLUSION: In early-stage CRC, the association of tumor side and effect on the time-to-recurrence and OS varies by stage. In stage III patients with an RP, poorer OS and cancer-specific survival outcomes are, in part, driven by inferior survival after recurrence, and tumor side did not influence adjuvant chemotherapy benefit.

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling.

The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.

A hybrid approach for automated mutation annotation of the extended human mutation landscape in scientific literature.

As the cost of DNA sequencing continues to fall, an increasing amount of information on human genetic variation is being produced that could help progress precision medicine. However, information about such mutations is typically first made available in the scientific literature, and is then later manually curated into more standardized genomic databases. This curation process is expensive, time-consuming and many variants do not end up being fully curated, if at all. Detecting mutations in the literature is the first key step towards automating this process. However, most of the current methods have focused on identifying mutations that follow existing nomenclatures. In this work, we show that there is a large number of mutations that are missed by using this standard approach. Furthermore, we implement the first mutation annotator to cover an extended mutation landscape, and we show that its F1 performance is the same performance as human annotation (F1 78.29 for manual annotation vs F1 79.56 for automatic annotation).

The efficient synthesis and purification of amyloid-ß(1-42) using an oligoethylene glycol-containing photocleavable lysine tag.

A short, monodisperse oligoethylene glycol-containing photocleavable lysine tag was developed to facilitate the efficient purification of hydrophobic and fibril-forming peptides. This new tag was used to prepare a modified Aß42 peptide with increased solubility and decreased propensity to aggregate in aqueous media. The solubilising tag was readily removed by irradiation with UV light and permitted the preparation and isolation of Aß42 in high purity and yield.

Toward hypoxia-selective rhenium and technetium tricarbonyl complexes.

With the aim of preparing hypoxia-selective imaging and therapeutic agents, technetium(I) and rhenium(I) tricarbonyl complexes with pyridylhydrazone, dipyridylamine, and pyridylaminocarboxylate ligands containing nitrobenzyl or nitroimidazole functional groups have been prepared. The rhenium tricarbonyl complexes were synthesized with short reaction times using microwave irradiation. Rhenium tricarbonyl complexes with deprotonated p-nitrophenyl pyridylhydrazone ligands are luminescent, and this has been used to track their uptake in HeLa cells using confocal fluorescent microscopy. Selected rhenium tricarbonyl complexes displayed higher uptake in hypoxic cells when compared to normoxic cells. A (99m)Tc tricarbonyl complex with a dipyridylamine ligand bearing a nitroimidazole functional group is stable in human serum and was shown to localize in a human renal cell carcinoma (RCC; SK-RC-52) tumor in a mouse.

Single-chain antibody conjugated to a cage amine chelator and labeled with positron-emitting copper-64 for diagnostic imaging of activated platelets.

Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.

Copper and silver complexes of tris(triazole)amine and tris(benzimidazole)amine ligands: evidence that catalysis of an azide-alkyne cycloaddition ("click") reaction by a silver tris(triazole)amine complex arises from copper impurities.

The synthesis and characterization of a silver complex of the tripodal triazole ligand, tris(benzyltriazolylmethyl)amine (TBTA, L(1)), that is used as promoter to enhance Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions is reported. X-ray analysis of the silver(I) complex with L(1) reveals a dinuclear cation, [Ag2(L(1))2](2+), that is essentially isostructural to the copper(I) analogue. While the [Ag2(L(1))2](BF4)2 complex provides catalysis for the azide-alkyne cycloaddition process, evidence is presented that this arises from trace copper contamination. The synthesis of silver(I), copper(II), and copper(I) complexes of a second tripodal ligand, tris(2-benzimidazolymethyl)amine (L(2)), which is used to enhance the rate of CuAAC reactions, is also reported. X-ray crystallography of the Cu(I) complex [Cu(I)3(L(2))2(CH3CN)2](BF4)3 offers structural insight into previous mechanistic speculation about the role of this ligand in the CuAAC reaction.

Conjugation of transferrin to azide-modified CdSe/ZnS core-shell quantum dots using cyclooctyne click chemistry.

Twinkle twinkle quantum dot: Conjugation of biomolecules to azide-modified quantum dots (QDs) through a bifunctional linker, using strain-promoted azide-alkyne cycloaddition with the QD and a squaramide linkage to the biomolecule (see scheme). Transferrin-conjugated QDs were internalized by transferrin-receptor expressing HeLa cells.

Cellular up-regulation of Nedd4 family interacting protein 1 (Ndfip1) using low levels of bioactive cobalt complexes.

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.

Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes.

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

PI(3,4,5)P3 Interactome.

Immobilizing chemically synthesized analogues of PI(3,4,5)P3 onto Affi-10 beads and incorporating them into liposomes allowed their use as affinity absorbents in the comprehensive analysis of the phosphoinositide interactome using cytosolic cell extracts of the LIM1215 colon cancer cell line. This led to the identification of 282 proteins that either interact with PI(3,4,5)P3 or are indirectly captured as part of a complex containing a PI(3,4,5)P3 binding partner. Identification of the proteins was achieved using affinity/LC-MS/MS experiments.

The PI(3,5)P2 and PI(4,5)P2 interactomes.

A comprehensive analysis of the phosphoinositide interactome has been performed using analogues of PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids which were immobilized onto Affi-10 beads or incorporated into liposomes for use as affinity absorbents with cytosolic extracts from colonic carcinoma cell lines. Affinity/LC/MS/MS experiments allowed identification of 388 proteins/protein complexes that appeared to interact specifically with the phosphoinositide targets: a number of novel potential phosphoinositide interacting proteins have been identified.