Automated VOI Analysis in FDDNP PET Using Structural Warping: Validation through Classification of Alzheimer's Disease Patients

We evaluate an automated approach to the cortical surface mapping (CSM) method of VOI analysis in PET. Although CSM has been previously shown to be successful, the process can be long and tedious. Here, we present an approach that removes these difficulties through the use of 3D image warping to a common space. We test this automated method using studies of FDDNP PET in Alzheimer's disease and mild cognitive impairment. For each subject, VOIs were created, through CSM, to extract regional PET data. After warping to the common space, a single set of CSM-generated VOIs was used to extract PET data from all subjects. The data extracted using a single set of VOIs outperformed the manual approach in classifying AD patients from MCIs and controls. This suggests that this automated method can remove variance in measurements of PET data and can facilitate accurate, high-throughput image analysis

Heat-induced-radiolabeling and click chemistry: A powerful combination for generating multifunctional nanomaterials

A key advantage of nanomaterials for biomedical applications is their ability to feature multiple small reporter groups (multimodality), or combinations of reporter groups and therapeutic agents (multifunctionality), while being targeted to cell surface receptors. Here a facile combination of techniques for the syntheses of multimodal, targeted nanoparticles (NPs) is presented, whereby heat-induced-radiolabeling (HIR) labels NPs with radiometals and so-called click chemistry is used to attach bioactive groups to the NP surface. Click-reactive alkyne or azide groups were first attached to the nonradioactive clinical Feraheme (FH) NPs. Resulting “Alkyne-FH” and “Azide-FH” intermediates, like the parent NP, tolerated 89Zr labeling by the HIR method previously described. Subsequently, biomolecules were quickly conjugated to the radioactive NPs by either copper-catalyzed or copper-free click reactions with high efficiency. Synthesis of the Alkyne-FH or Azide-FH intermediates, followed by HIR and then by click reactions for biomolecule attachment, provides a simple and potentially general path for the synthesis of multimodal, multifunctional, and targeted NPs for biomedical applications

Outline of heat induced radiolabeling (HIR) and click chemistry surface functionalization used to obtain multimodal, targeted NPs.

<p>Our previous HIR of FH NPs (top) yielded non-surface functionalized, radioactive NPs (red core). The alkyne and azide functionalized FH intermediates were synthesized (Azide-FH, <b>4</b> and Alkyne-FH, <b>5</b>) and labeled by HIR reaction, to yield ⁸⁹Zr-Azide-FH (^<b>89</b><b>Zr-4</b>) and ⁸⁹Zr-Alkyne-FH (^<b>89</b><b>Zr-5</b>). Imaging detection modalities for the NPs are in bold. NPs targeted to folate receptors (⁸⁹Zr-Folate-FH, ^<b>89</b><b>Zr-11</b>), integrins (RGD-FH, <b>14</b>) or NPs with protamines (⁸⁹Zr-Cy5.5-Protamine-FH, ^<b>89</b><b>Zr-16</b>) were then synthesized. Detailed synthetic schemes are given in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.g002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.g004" target="_blank">4</a>.</p

Surface functionalization of Feraheme (FH, 1) with azide or alkyne groups and radiolabeling functionalized NPs by HIR.

<p>a) Using FH (<b>1</b>)<b>,</b> the Azide-FH (<b>4</b>) and Alkyne-FH (<b>5</b>) were synthesized. Portions of Azide-FH <b>(4)</b> and Alkyne-FH (<b>5</b>) were then radiolabeled by HIR, yielding ⁸⁹Zr-Azide-FH (^<b>89</b><b>Zr-4</b>) and ⁸⁹Zr-Alkyne-FH (^<b>89</b><b>Zr-5</b>). To determine reactive azide or reactive alkynes, NPs were reacted with the appropriate click reactive Cy5.5 fluorochromes, with Cy5.5s shown as the yellow stars of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.g001" target="_blank">Fig 1</a>. After removal of the unreacted Cy5.5s (DBCO-Cy5.5, <b>6</b> or Azide-Cy5.5, <b>7</b>), the number of Cy5.5’s per NP was determined from absorption spectra examples of which are shown in Fig 2b–2e. Controls for covalent binding were a reaction of FH (<b>1</b>) and DBCO-Cy5.5 (<b>6</b>) and a reaction of Azide-FH <b>(4)</b> and DBCO-Cy5.5 (<b>6</b>) preoccupied with DBCO-NH₂. Values in parentheses are the numbers of reactive groups per NP with values summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.t001" target="_blank">Table 1</a>.</p

Summary of HIR NP’s and Surface Functionalization Reactions.

<p>Summary of HIR NP’s and Surface Functionalization Reactions.</p

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis.

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer's disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease

Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C’s renal clearance ability and Feraheme’s FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation

Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

Summary: It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals. : Argus et al. developed Fatty Acid Source Analysis (FASA), a model that quantifies cellular fatty acid synthesis, elongation, and import. FASA is used to demonstrate that elongation can be a major contributor to cellular fatty acid content and that different stimuli reprogram macrophage fatty acid elongation pathways in distinct ways. Keywords: fatty acid homeostasis, fatty acid modeling, stable isotope labelin

Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling.

It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis

<div><p>PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of <i>PICALM</i>-deficient and <i>PICALM</i>-expressing cells. Pathway analysis demonstrated that <i>PICALM</i> expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in <i>PICALM</i>-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.</p></div