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ACS Infectious Diseases Dec 2019Bedaquiline is a promising drug against tuberculosis (TB), but limited data are available on its intralesional pharmacokinetics. Moreover, current techniques rely on...
Bedaquiline is a promising drug against tuberculosis (TB), but limited data are available on its intralesional pharmacokinetics. Moreover, current techniques rely on invasive tissue resection, which is difficult in humans and generally limited even in animals. In this study, we developed a novel radiosynthesis for Br-bedaquiline and performed noninvasive, longitudinal whole-body positron emission tomography (PET) in live, -infected mice over 48 h. After the intravenous injection, Br-bedaquiline distributed to all organs and selectively localized to adipose tissue and liver, with excellent penetration into infected lung lesions (86%) and measurable penetration into the brain parenchyma (15%). Ex vivo high resolution, two-dimensional autoradiography, and same section hematoxylin/eosin and immunofluorescence provided detailed intralesional drug biodistribution. PET bioimaging and high-resolution autoradiography are novel techniques that can provide detailed, multicompartment, and intralesional pharmacokinetics of new and existing TB drugs. These technologies can significantly advance efforts to optimize drug dosing.
Topics: Administration, Intravenous; Animals; Autoradiography; Diarylquinolines; Disease Models, Animal; Female; Humans; Lung; Mice; Positron-Emission Tomography; Tuberculosis; Whole Body Imaging
PubMed: 31345032
DOI: 10.1021/acsinfecdis.9b00207 -
Cold Spring Harbor Protocols Dec 2020Bands of radioactive DNA separated by polyacrylamide gel electrophoresis may be detected by autoradiography or phosphorimaging. Analytical polyacrylamide gels containing...
Bands of radioactive DNA separated by polyacrylamide gel electrophoresis may be detected by autoradiography or phosphorimaging. Analytical polyacrylamide gels containing radioactive DNA are usually fixed and dried before autoradiography. However, if bands of radioactive DNA are to be recovered from the gel, the gel should generally not be fixed or dried.
Topics: Acrylic Resins; Autoradiography; DNA; Electrophoresis, Polyacrylamide Gel; Isotope Labeling; Radiographic Image Enhancement; Radioisotopes; X-Ray Film
PubMed: 33262237
DOI: 10.1101/pdb.prot100420 -
Journal of Neuropathology and... Sep 2019The sudden infant death syndrome (SIDS) is the leading cause of postneonatal infant mortality in the United States today, with an overall rate of 0.39/1000 live births.... (Review)
Review
The sudden infant death syndrome (SIDS) is the leading cause of postneonatal infant mortality in the United States today, with an overall rate of 0.39/1000 live births. It is defined as the sudden and unexpected death of an infant <12 months of age that remains unexplained after a complete autopsy, death scene investigation, and review of the clinical history. The serotonin brainstem hypothesis has been a leading hypothesis for SIDS over the last 2 decades. Our laboratory has studied this hypothesis over time with a variety of tissue techniques, including tissue receptor autoradiography, high performance liquid chromatography, Western blot analysis, immunocytochemistry, and proteomics. The purpose of this article is to review the progress in our laboratory toward supporting this hypothesis. We conclude that an important subset of SIDS infants has serotonergic abnormalities resulting from a "core lesion" in the medullary reticular formation comprised of nuclei that contain serotonin neurons. This lesion could lead to a failure of protective brainstem responses to homeostatic challenges during sleep in a critical developmental period which cause sleep-related sudden death.
Topics: Brain Stem; Humans; Infant; Infant, Newborn; Midbrain Reticular Formation; Serotonergic Neurons; Serotonin; Sudden Infant Death
PubMed: 31397480
DOI: 10.1093/jnen/nlz062 -
Journal of Alzheimer's Disease : JAD 2024Although sporadic Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology, familial AD is associated with specific gene mutations. A commonality...
BACKGROUND
Although sporadic Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology, familial AD is associated with specific gene mutations. A commonality between these forms of AD is that both display multiple pathogenic events including cholinergic and lipid dysregulation.
OBJECTIVE
We aimed to identify the relevant lipids and the activity of their related receptors in the frontal cortex and correlating them with cognition during the progression of AD.
METHODS
MALDI-mass spectrometry imaging (MSI) and functional autoradiography was used to evaluate the distribution of phospholipids/sphingolipids and the activity of cannabinoid 1 (CB1), sphingosine 1-phosphate 1 (S1P1), and muscarinic M2/M4 receptors in the frontal cortex (FC) of people that come to autopsy with premortem clinical diagnosis of AD, mild cognitive impairment (MCI), and no cognitive impairment (NCI).
RESULTS
MALDI-MSI revealed an increase in myelin-related lipids, such as diacylglycerol (DG) 36:1, DG 38:5, and phosphatidic acid (PA) 40:6 in the white matter (WM) in MCI compared to NCI, and a downregulation of WM phosphatidylinositol (PI) 38:4 and PI 38:5 levels in AD compared to NCI. Elevated levels of phosphatidylcholine (PC) 32:1, PC 34:0, and sphingomyelin 38:1 were observed in discrete lipid accumulations in the FC supragranular layers during disease progression. Muscarinic M2/M4 receptor activation in layers V-VI decreased in AD compared to MCI. CB1 receptor activity was upregulated in layers V-VI, while S1P1 was downregulated within WM in AD relative to NCI.
CONCLUSIONS
FC WM lipidomic alterations are associated with myelin dyshomeostasis in prodromal AD, suggesting WM lipid maintenance as a potential therapeutic target for dementia.
Topics: Humans; Alzheimer Disease; Cognitive Dysfunction; Receptor, Muscarinic M4; Frontal Lobe; Cholinergic Agents; Lipids
PubMed: 38578893
DOI: 10.3233/JAD-231485 -
NeuroImage Dec 2022Neurotransmitter receptors modulate signaling between neurons. Thus, neurotransmitter receptors and transporters play a key role in shaping brain function. Due to the...
Neurotransmitter receptors modulate signaling between neurons. Thus, neurotransmitter receptors and transporters play a key role in shaping brain function. Due to the lack of comprehensive neurotransmitter receptor/transporter density datasets, microarray gene expression measuring mRNA transcripts is often used as a proxy for receptor densities. In the present report, we comprehensively test the spatial correlation between gene expression and protein density for a total of 27 neurotransmitter receptors, receptor binding-sites, and transporters across 9 different neurotransmitter systems, using both PET and autoradiography radioligand-based imaging modalities. We find poor spatial correspondences between gene expression and density for all neurotransmitter receptors and transporters except four single-protein metabotropic receptors (5-HT, CB, D, and MOR). These expression-density associations are related to gene differential stability and can vary between cortical and subcortical structures. Altogether, we recommend using direct measures of receptor and transporter density when relating neurotransmitter systems to brain structure and function.
Topics: Humans; Receptors, Neurotransmitter; Brain; Autoradiography; Neurotransmitter Agents; Carrier Proteins; Gene Expression
PubMed: 36209794
DOI: 10.1016/j.neuroimage.2022.119671 -
European Journal of Nuclear Medicine... Jan 2022The deposition of β-amyloid (Aβ) in the brain is a biomarker of Alzheimer's disease (AD). Highly sensitive Aβ positron emission tomography (PET) imaging plays an...
BACKGROUND
The deposition of β-amyloid (Aβ) in the brain is a biomarker of Alzheimer's disease (AD). Highly sensitive Aβ positron emission tomography (PET) imaging plays an essential role in diagnosing and evaluating the therapeutic effects of AD.
AIM
To synthesize a new Aβ tracer [F]DRKXH1 (5-(4-(6-(2-[18]fluoroethoxy)ethoxy)imidazo[1,2-alpha]pyridin-2-yl)phenyl) and evaluate the tracer performance by biodistribution analysis, in vivo small-animal PET-CT dynamic scan, ex vivo and in vitro autoradiography, and PET in human subjects.
METHODS
[F]DRKXH1 was synthesized automatically by the GE FN module. Log D (pH 7.4) and biodistribution of [F]DRKXH1 were investigated. Small-animal-PET was used for [F]DRKXH1 and [F]AV45 imaging study in AD transgenic mice (APPswe/PSEN1dE9) and age-matched normal mice. The distribution volume ratios (DVR) and standardized uptake value ratios (SUVRs) were calculated with the cerebellum as the reference region. The deposition of Aβ plaques in the brain of AD transgenic mice was determined by ex vivo autoradiography and immunohistochemistry. In vitro autoradiography was performed in the postmortem brain sections of AD patients and healthy controls. Two healthy control subjects and one AD patient was subjected to in vivo PET study using [F]DRKXH1.
RESULTS
The yield of [F]DRKXH1 was 40%, and the specific activity was 156.64 ± 11.55 GBq/μmol. [F]DRKXH1 was mainly excreted through the liver and kidney. The small-animal PET study showed high initial brain uptake and rapid washout of [F]DRKXH1. The concentration of [F]DRKXH1 was detected in the cortex and hippocampus of AD transgenic mice brain. The cortex DVR of AD transgenic mice was higher than that of WT mice (P < 0.0001). Moreover, the SUVRs of AD transgenic mice were higher than those of WT mice based on the 0-60-min dynamic scanning. In vitro autoradiography showed a significant concentration of tracer in the Aβ plaque-rich areas in the brain of AD transgenic mice. The DVR value of [F]-DRKXH1 is higher than that of [F]-AV45 (1.29 ± 0.05 vs. 1.05 ± 0.08; t = 5.33, P = 0.0003). Autoradiography of postmortem human brain sections showed [F]DRKXH1-labeled Aβ plaques in the AD brain. The AD patients had high retention in cortical regions, while healthy control subjects had uniformly low radioactivity uptake.
CONCLUSIONS
[F]DRKXH1 is an Aβ tracer with high sensitivity in preclinical study and has the potential for in vivo detection of the human brain.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Aniline Compounds; Animals; Brain; Humans; Mice; Mice, Transgenic; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Tissue Distribution
PubMed: 34292345
DOI: 10.1007/s00259-021-05421-0 -
Methods in Molecular Biology (Clifton,... 2022The structure of an RNA molecule is often critical for its correct functioning, post-transcriptional regulation, and/or translation. Predicting RNA secondary structures...
The structure of an RNA molecule is often critical for its correct functioning, post-transcriptional regulation, and/or translation. Predicting RNA secondary structures with in silico tools is relatively straightforward with the large array of software and webservers available. However, for long RNAs and RNA at high temperatures, in silico predictions are less accurate and require experimental validation. To this end, a variety of structural probing reagents are commonly used, both for in vitro and in vivo mapping of RNA structure. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments make use of a nonbase-specific modifying reagent, acylating all conformationally flexible (mainly single-stranded or unpaired) nucleotides and have been employed both for in vitro and in vivo modification of RNA. Here, we describe a protocol for an in vitro SHAPE experiment, starting from in vitro transcribed RNA using a T7 polymerase system. This RNA is folded and subsequently modified in vitro with the SHAPE-reagent N-methyl isatoic anhydride (NMIA). Primer extension employing a radioactive P-labeled primer that binds the RNA downstream of the structure of interest will generate cDNA until the reverse transcriptase enzyme is halted by the introduced SHAPE modifications. Denaturing acrylamide gel electrophoresis of the pool of P-labeled cDNAs and the corresponding sequencing ladders, followed by autoradiography, will expose these stops in reverse transcription (RT) and will therefore enable to identify single-stranded nucleotides in the RNA of interest. These RT stops and NMIA-modification efficiencies can be quantified with ImageJ software and can be used to validate or increase the accuracy of RNA secondary structure predictions.
Topics: Acylation; Nucleic Acid Conformation; Nucleotides; RNA; RNA-Directed DNA Polymerase; Sequence Analysis, RNA; Software
PubMed: 35922631
DOI: 10.1007/978-1-0716-2413-5_14 -
Molecular Imaging 2020Positron-emitting nuclides have long been used as imaging agents in medical science to spatially trace processes non-invasively, allowing for real-time molecular imaging... (Review)
Review
Positron-emitting nuclides have long been used as imaging agents in medical science to spatially trace processes non-invasively, allowing for real-time molecular imaging using low tracer concentrations. This ability to non-destructively visualize processes in real time also makes positron imaging uniquely suitable for probing various processes in plants and porous environmental media, such as soils and sediments. Here, we provide an overview of historical and current applications of positron imaging in environmental research. We highlight plant physiological research, where positron imaging has been used extensively to image dynamics of macronutrients, signalling molecules, trace elements, and contaminant metals under various conditions and perturbations. We describe how positron imaging is used in porous soils and sediments to visualize transport, flow, and microbial metabolic processes. We also address the interface between positron imaging and other imaging approaches, and present accompanying chemical analysis of labelled compounds for reviewed topics, highlighting the bridge between positron imaging and complementary techniques across scales. Finally, we discuss possible future applications of positron imaging and its potential as a nexus of interdisciplinary biogeochemical research.
Topics: Electrons; Plants; Radioactive Tracers; Soil
PubMed: 33119419
DOI: 10.1177/1536012120966405 -
European Journal of Nuclear Medicine... Jun 2021Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to...
PURPOSE
Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to detect pathological aggregates of αSYN remains lacking. Here, we report the development of a positron emission tomography (PET) tracer based on anle138b, a compound shown to have therapeutic activity in animal models of neurodegenerative diseases.
METHODS
Specificity and selectivity of [H]MODAG-001 were tested in in vitro binding assays using recombinant fibrils. After carbon-11 radiolabeling, the pharmacokinetic and metabolic profile was determined in mice. Specific binding was quantified in rats, inoculated with αSYN fibrils and using in vitro autoradiography in human brain sections of Lewy body dementia (LBD) cases provided by the Neurobiobank Munich (NBM).
RESULTS
[H]MODAG-001 revealed a very high affinity towards pure αSYN fibrils (K = 0.6 ± 0.1 nM) and only a moderate affinity to hTau46 fibrils (K = 19 ± 6.4 nM) as well as amyloid-β fibrils (K = 20 ± 10 nM). [C]MODAG-001 showed an excellent ability to penetrate the mouse brain. Metabolic degradation was present, but the stability of the parent compound improved after selective deuteration of the precursor. (d)-[C]MODAG-001 binding was confirmed in fibril-inoculated rat striata using in vivo PET imaging. In vitro autoradiography showed no detectable binding to aggregated αSYN in human brain sections of LBD cases, most likely, because of the low abundance of aggregated αSYN against background protein.
CONCLUSION
MODAG-001 provides a promising lead structure for future compound development as it combines a high affinity and good selectivity in fibril-binding assays with suitable pharmacokinetics and biodistribution properties.
Topics: Animals; Carbon Radioisotopes; Lewy Body Disease; Mice; Neurodegenerative Diseases; Positron-Emission Tomography; Radiopharmaceuticals; Rats; Tissue Distribution; alpha-Synuclein
PubMed: 33369690
DOI: 10.1007/s00259-020-05133-x -
Molecular Pharmaceutics Nov 2022Noninvasively monitoring activated fibroblasts is of great value for understanding the dynamic process of myocardial fibrosis after myocardial infarction (MI). This...
Noninvasively monitoring activated fibroblasts is of great value for understanding the dynamic process of myocardial fibrosis after myocardial infarction (MI). This study aimed to evaluate the feasibility of Ga-labeled fibroblast activation protein inhibitor 04 (Ga-FAPI-04) for monitoring reparative fibrosis and reactive fibrosis after MI. MI models were prepared by ligation of the left anterior descending (LAD) coronary artery and validated by electrocardiogram and F-FDG PET/CT 1 day after MI and hematoxylin and eosin (HE) staining. Ga-FAPI-04 PET/CT scans (1, 3, 6, 9, 12, 15, 18, 21, 28, and 35 days after MI) were carried out in MI rats and sham-operated rats without ligation of LAD. Blocking experiments were carried out on MI rats on day 7 after MI with Ga-FAPI-04 and excessive FAPI-04. Autoradiography, HE staining, Masson's trichrome staining, and immunofluorescence staining were carried out for validation. The infarcted area with decreased or defective myocardial metabolic activity in F-FDG PET/CT correspondingly showed high Ga-FAPI-04 uptake in the MI rats. The myocardial tracer uptake was significantly different between MI and sham-operated rats from day 1 to 28 after MI and reached peak value 6 days after MI (0.806 ± 0.257%ID/cc vs 0.199 ± 0.012%ID/cc, < 0.05). Tracer uptake at the infarcted myocardium and normal tissues in MI rats decreased significantly after blocking. Obvious tracer uptake was confirmed by autoradiography, and immunofluorescence staining showed FAP+ cells in the infarcted myocardium and border zone. Masson's trichrome staining of the heart sections of MI rats at different times suggested the presence of myocardial fibrosis. Ga-FAPI-04 uptake was not observed in the distal uninjured myocardium throughout the observation period. In conclusion, Ga-FAPI-04 PET could noninvasively monitor the activated fibroblasts in the early stage post acute MI and may be helpful for evaluating the degree of reparative fibrosis, while reactive fibrosis monitoring still needs further study.
Topics: Animals; Rats; Fibrosis; Fluorodeoxyglucose F18; Gallium Radioisotopes; Myocardial Infarction; Positron Emission Tomography Computed Tomography; Quinolines
PubMed: 35969029
DOI: 10.1021/acs.molpharmaceut.2c00551