Image-Derived Input Function for Human Brain Using High Resolution PET Imaging with [11C](R)-rolipram and [11C]PBR28

Background

The aim of this study was to test seven previously published image-input methods in state-of-the-art high resolution PET brain images. Images were obtained with a High Resolution Research Tomograph plus a resolution-recovery reconstruction algorithm using two different radioligands with different radiometabolite fractions. Three of the methods required arterial blood samples to scale the image-input, and four were blood-free methods.

Methods

All seven methods were tested on twelve scans with [¹¹C](R)-rolipram, which has a low radiometabolite fraction, and on nineteen scans with [¹¹C]PBR28 (high radiometabolite fraction). Logan V _T values for both blood and image inputs were calculated using the metabolite-corrected input functions. The agreement of image-derived Logan V _T values with the reference blood-derived Logan V _T values was quantified using a scoring system. Using the image input methods that gave the most accurate results with Logan analysis, we also performed kinetic modelling with a two-tissue compartment model.

Results

For both radioligands the highest scores were obtained with two blood-based methods, while the blood-free methods generally performed poorly. All methods gave higher scores with [¹¹C](R)-rolipram, which has a lower metabolite fraction. Compartment modeling gave less reliable results, especially for the estimation of individual rate constants.

Conclusion

Our study shows that: 1) Image input methods that are validated for a specific tracer and a specific machine may not perform equally well in a different setting; 2) despite the use of high resolution PET images, blood samples are still necessary to obtain a reliable image input function; 3) the accuracy of image input may also vary between radioligands depending on the magnitude of the radiometabolite fraction: the higher the metabolite fraction of a given tracer (e.g., [¹¹C]PBR28), the more difficult it is to obtain a reliable image-derived input function; and 4) in association with image inputs, graphical analyses should be preferred over compartmental modelling.     

Imaging Amyloid Fibrils within Cells Using a Se-Labelling Strategy

The process of aggregation leading to amyloid formation by peptides and proteins is associated with diseases ranging from systemic amyloidoses to neurodegenerative disorders such as Alzheimer's disease. A key question in understanding the link between amyloid formation and its pathological consequences is the ultrastructural localisation and morphological form of amyloid species within the cellular environment. The acquisition of such information has proven to be challenging, but we report here a novel approach that enables amyloid fibrils to be visualised directly within a cell. First, fibrils are assembled from selenium analogues of the sulfur-containing cysteine peptides, and then, atomic number contrast transmission electron microscopy is used to detect the selenium doped species selectively within the carbon-rich background of the cell. We demonstrate the power of this approach by imaging human monocyte-derived macrophage cells that have been exposed to fibrils from an amyloidogenic fragment of the disease-associated protein transthyretin. The ready incorporation of seleno-cysteine and methionine instead of their natural sulfur-containing analogues, a feature that is already commonly used in X-ray diffraction studies of proteins, suggests that this method can be used as a general strategy to image specific peptides and proteins within the cellular environment using electron microscopy.     

Exploration of Insulin Amyloid Polymorphism Using Raman Spectroscopy and Imaging

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na₂SO₄) by heating at 60, 70, 80, and 90°C. The intensity ratio of two Raman bands at 1672 and 1657 cm⁻¹ due to antiparallel β-sheet and α-helix structures, respectively, was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the β-sheet structure was higher in the case with salts, especially at a higher temperature with Na₂SO₄. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm⁻¹) and the ratio of the tyrosine doublet I₈₅₀/I₈₂₆ were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis. Especially, amyloid fibrils with Na₂SO₄ media formed the gauche-gauche-gauche conformation of disulfide bond at a higher rate, but without any salts, the gauche-gauche-gauche conformation was partially transformed into the gauche-gauche-trans conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three- dimensional structural differences of amyloid polymorphisms. These results demonstrate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.     

DCE-MRI of the Liver: Reconstruction of the Arterial Input Function Using a Low Dose Pre-Bolus Contrast Injection

Purpose

To assess the quality of the arterial input function (AIF) reconstructed using a dedicated pre-bolus low-dose contrast material injection imaged with a high temporal resolution and the resulting estimated liver perfusion parameters.

Materials and Methods

In this IRB–approved prospective study, 24 DCE-MRI examinations were performed in 21 patients with liver disease (M/F 17/4, mean age 56 y). The examination consisted of 1.3 mL and 0.05 mmol/kg of gadobenate dimeglumine for pre-bolus and main bolus acquisitions, respectively. The concentration-curve of the abdominal aorta in the pre-bolus acquisition was used to reconstruct the AIF. AIF quality and shape parameters obtained with pre-bolus and main bolus acquisitions and the resulting estimated hepatic perfusion parameters obtained with a dual-input single compartment model were compared between the 2 methods. Test–retest reproducibility of perfusion parameters were assessed in three patients.

Results

The quality of the pre-bolus AIF curve was significantly better than that of main bolus AIF. Shape parameters peak concentration, area under the time activity curve of gadolinium contrast at 60 s and upslope of pre-bolus AIF were all significantly higher, while full width at half maximum was significantly lower than shape parameters of main bolus AIF. Improved liver perfusion parameter reproducibility was observed using pre-bolus acquisition [coefficient of variation (CV) of 4.2%–38.7% for pre-bolus vs. 12.1–71.4% for main bolus] with the exception of distribution volume (CV of 23.6% for pre-bolus vs. 15.8% for main bolus). The CVs between pre-bolus and main bolus for the perfusion parameters were lower than 14%.

Conclusion

The AIF reconstructed with pre-bolus low dose contrast injection displays better quality and shape parameters and enables improved liver perfusion parameter reproducibility, although the resulting liver perfusion parameters demonstrated no clinically significant differences between pre-bolus and main bolus acquisitions.     

Brain Characterization Using Normalized Quantitative Magnetic Resonance Imaging

Objectives

To present a method for generating reference maps of typical brain characteristics of groups of subjects using a novel combination of rapid quantitative Magnetic Resonance Imaging (qMRI) and brain normalization. The reference maps can be used to detect significant tissue differences in patients, both locally and globally.

Materials and Methods

A rapid qMRI method was used to obtain the longitudinal relaxation rate (R₁), the transverse relaxation rate (R₂) and the proton density (PD). These three tissue properties were measured in the brains of 32 healthy subjects and in one patient diagnosed with Multiple Sclerosis (MS). The maps were normalized to a standard brain template using a linear affine registration. The differences of the mean value ofR₁, R₂ and PD of 31 healthy subjects in comparison to the oldest healthy subject and in comparison to an MS patient were calculated. Larger anatomical structures were characterized using a standard atlas. The vector sum of the normalized differences was used to show significant tissue differences.

Results

The coefficient of variation of the reference maps was high at the edges of the brain and the ventricles, moderate in the cortical grey matter and low in white matter and the deep grey matter structures. The elderly subject mainly showed significantly lower R₁ and R₂ and higher PD values along all sulci. The MS patient showed significantly lower R₁ and R₂ and higher PD values at the edges of the ventricular system as well as throughout the periventricular white matter, at the internal and external capsules and at each of the MS lesions.

Conclusion

Brain normalization of rapid qMRI is a promising new method to generate reference maps of typical brain characteristics and to automatically detect deviating tissue properties in the brain.     

Diaphragm Postural Function Analysis Using Magnetic Resonance Imaging

We present a postural analysis of diaphragm function using magnetic resonance imaging (MRI). The main aim of the study was to identify changes in diaphragm motion and shape when postural demands on the body were increased (loading applied to a distal part of the extended lower extremities against the flexion of the hips was used). Sixteen healthy subjects were compared with 17 subjects suffering from chronic low back pain and in whom structural spine disorders had been identified. Two sets of features were calculated from MRI recordings: dynamic parameters reflecting diaphragm action, and static parameters reflecting diaphragm anatomic characteristics. A statistical analysis showed that the diaphragm respiratory and postural changes were significantly slower, bigger in size and better balanced in the control group. When a load was applied to the lower limbs, the pathological subjects were mostly not able to maintain the respiratory diaphragm function, which was lowered significantly. Subjects from the control group showed more stable parameters of both respiratory and postural function. Our findings consistently affirmed worse muscle cooperation in the low back pain population subgroup. A clear relation with spinal findings and with low back pain remains undecided, but various findings in the literature were confirmed. The most important finding is the need to further address various mechanisms used by patients to compensate deep muscle insufficiency.     

Qualitative and Quantitative Analysis of DNA Fragmentation Using Digital Imaging

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-α, transforming growth factor-β1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.     

Spatiotemporal Characterization of a Fibrin Clot Using Quantitative Phase Imaging

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.     

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death ¹. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection ^2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature ⁴.Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin ^5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology ^7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin ⁹, allowed breakthroughs in the field.In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma ^10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.     

The Amyloid Precursor Protein Controls PIKfyve Function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP''s implication in Alzheimer''s disease. Using our recently developed proteo-liposome assay we established the interactome of APP''s intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer''s disease.     

Quantitative Assessment of Murine Articular Cartilage and Bone Using X-Ray Phase-Contrast Imaging

Murine models for rheumatoid arthritis (RA) research can provide important insights for understanding RA pathogenesis and evaluating the efficacy of novel treatments. However, simultaneously imaging both murine articular cartilage and subchondral bone using conventional techniques is challenging because of low spatial resolution and poor soft tissue contrast. X-ray phase-contrast imaging (XPCI) is a new technique that offers high spatial resolution for the visualisation of cartilage and skeletal tissues. The purpose of this study was to utilise XPCI to observe articular cartilage and subchondral bone in a collagen-induced arthritis (CIA) murine model and quantitatively assess changes in the joint microstructure. XPCI was performed on the two treatment groups (the control group and CIA group, n = 9 per group) to monitor the progression of damage to the femur from the knee joint in a longitudinal study (at 0, 4 and 8 weeks after primary injection). For quantitative assessment, morphologic parameters were measured in three-dimensional (3D) images using appropriate image analysis software. Our results showed that the average femoral cartilage volume, surface area and thickness were significantly decreased (P<0.05) in the CIA group compared to the control group. Meanwhile, these decreases were accompanied by obvious destruction of the surface of subchondral bone and a loss of trabecular bone in the CIA group. This study confirms that XPCI technology has the ability to qualitatively and quantitatively evaluate microstructural changes in mouse joints. This technique has the potential to become a routine analysis method for accurately monitoring joint damage and comprehensively assessing treatment efficacy.     

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.     

Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.     

AL Amyloid Imaging and Therapy with a Monoclonal Antibody to a Cryptic Epitope on Amyloid Fibrils

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K_D of ∼10 nM. Binding was inhibited in the presence of the –Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.     

Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aβ (TMR-Aβ) to characterize its stability and heterogeneity. Disaggregation of TMR-Aβ amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aβ amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.     

Imaging Lung Function in Mice Using SPECT/CT and Per-Voxel Analysis

Chronic lung disease is a major worldwide health concern but better tools are required to understand the underlying pathologies. Ventilation/perfusion (V/Q) single photon emission computed tomography (SPECT) with per-voxel analysis allows for non-invasive measurement of regional lung function. A clinically adapted V/Q methodology was used in healthy mice to investigate V/Q relationships. Twelve week-old mice were imaged to describe normal lung function while 36 week-old mice were imaged to determine how age affects V/Q. Mice were ventilated with Technegas™ and injected with ^99mTc-macroaggregated albumin to trace ventilation and perfusion, respectively. For both processes, SPECT and CT images were acquired, co-registered, and quantitatively analyzed. On a per-voxel basis, ventilation and perfusion were moderately correlated (R = 0.58±0.03) in 12 week old animals and a mean log(V/Q) ratio of −0.07±0.01 and standard deviation of 0.36±0.02 were found, defining the extent of V/Q matching. In contrast, 36 week old animals had significantly increased levels of V/Q mismatching throughout the periphery of the lung. Measures of V/Q were consistent across healthy animals and differences were observed with age demonstrating the capability of this technique in quantifying lung function. Per-voxel analysis and the ability to non-invasively assess lung function will aid in the investigation of chronic lung disease models and drug efficacy studies.     

Imaging Flow Cytometry Illuminates New Dimensions of Amyloid Peptide-Membrane Interactions

Membrane interactions of amyloidogenic proteins constitute central determinants both in protein aggregation as well as in amyloid cytotoxicity. Most reported studies of amyloid peptide-membrane interactions have employed model membrane systems combined with application of spectroscopy methods or microscopy analysis of individual binding events. Here, we applied for the first time, to our knowledge, imaging flow cytometry for investigating interactions of representative amyloidogenic peptides, namely, the 106–126 fragment of prion protein (PrP(106–126)) and the human islet amyloid polypeptide (hIAPP), with giant lipid vesicles. Imaging flow cytometry was also applied to examine the inhibition of PrP(106–126)-membrane interactions by epigallocatechin gallate, a known modulator of amyloid peptide aggregation. We show that imaging flow cytometry provided comprehensive population-based statistical information upon morphology changes of the vesicles induced by PrP(106–126) and hIAPP. Specifically, the experiments reveal that both PrP(106–126) and hIAPP induced dramatic transformations of the vesicles, specifically disruption of the spherical shapes, reduction of vesicle circularity, lobe formation, and modulation of vesicle compactness. Interesting differences, however, were apparent between the impact of the two peptides upon the model membranes. The morphology analysis also showed that epigallocatechin gallate ameliorated vesicle disruption by PrP(106–126). Overall, this study demonstrates that imaging flow cytometry provides powerful means for disclosing population-based morphological membrane transformations induced by amyloidogenic peptides and their inhibition by aggregation modulators.     

Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.     

Distinguishing Closely Related Amyloid Precursors Using an RNA Aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β₂-microglobulin (hβ₂m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ₂m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ₂m with an EC₅₀ of ∼200 nm. NMR spectroscopy was used to assign the ¹H-¹⁵N HSQC spectrum of the B6-hβ₂m complex, revealing that the aptamer binds to the face of hβ₂m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ₂m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.     

血清淀粉样P物质的结构、表达调控及功能

血清淀粉样Ｐ物质 (ｓｅｒｕｍａｍｙｌｏｉｄＰｃｏｍ ｐｏｎｅｎｔ,ＳＡＰ) ,作为一种古老的蛋白质 ,与Ｃ反应蛋白 (ＣＲＰ)构成血清正五聚蛋白家族 ,其系统发生学非常保守 ,表明具有重要生理功能。虽然它广泛存在于脊椎动物 ,并且证明与机体淀粉样病变有关 ,但它的主要正常功能尚不清楚。近年来 ,随着分子生物学技术的发展 ,对其三维结构、结合配体及与疾病的关系等方面展开了较为深入细致的研究。本文就这一领域的研究进展作一综述。1 .ＳＡＰ的分子结构ＳＡＰ为血清正常糖蛋白 ,与经典的ＣＲＰ构成Ｃａ2 依赖性配体结合的血清正五聚…