Nonspecific Signal Research Articles

The GEM-handle as convenient labeling strategy for bimodal single-domain antibody-based tracers carrying 99mTc and a near-infrared fluorescent dye for intra-operative decision-making.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99mTc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99mTc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy

BackgroundEarly infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. MethodsFour families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. ResultsWe present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). ConclusionsOur results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.

B-087 The Antifouling Properties of BSA on Magnetic Beads are Affected by BSA Grade, Bead Type, and pH

Abstract Background BSA (bovine serum albumin) is a protein that is widely used for its antifouling properties in a variety of immunoassays. One such immunoassay is a bead-based ELISA (enzyme-linked immunosorbent assay), which can provide quantitative detection of many diverse analytes. BSA increases the sensitivity of a bead-based ELISA by coating the beads and blocking nonspecific interactions, thereby lowering background noise. However, the BSA grade chosen for the assay is frequently based on experience, as there is a lack of understanding of how BSA grades interact with different bead surfaces under various assay conditions. The objective of this study is to examine if different BSA grades work better with particular bead chemistries and how pH influences those interactions. Specifically, the aim is to quantify the amount of BSA on ELISA beads and assess the blocking of nonspecific antibody signal with different bead types, BSA grades, and pH. Methods The mass of BSA binding to beads was measured by heating BSA-coated beads in 1x SDS loading dye, then running on a denaturing PAGE gel. Total protein was measured by densitometry against a standard curve using ImageJ. The amount of blocking was measured using a modified bead-based ELISA, comparing the amount of antibody-HRP binding to the beads in the presence and absence of BSA. The amount of antibody-HRP was measured using a SpectraMax Plus 384 plate reader against a standard curve. All data were analyzed using GraphPad Prism and presented as the average of at least three independent replicates with error bars representing standard deviation. Conditions tested include different Dynabeads (hydrophilic vs. hydrophobic - Dynabeads M-270 Carboxylic Acid and M-280 Tosylactivated), BSA grade (fatty acid (FA) vs. fatty acid-free (FAF) - Proliant SKUs 68100 and 68700), and pH (7 vs. 5.2). Results The choice of pH is more influential than the BSA grade on bead surface coating and blocking, regardless of bead type. Lowering the pH from 7 to 5.2 increases the mass of BSA on the beads ∼1.7-fold for hydrophobic beads and ∼10-fold for hydrophilic beads. However, the mass of BSA on the beads is reduced after the pH returns to 7, indicating the increased BSA binding at pH 5.2 is reversible. We also observed BSA decreases nonspecific antibody-bead interactions by up to 90%, depending on assay conditions. Though the amount of BSA coating the beads between FA and FAF grade BSA is not significant, FAF grade BSA blocks the antibody-bead interactions ∼10% more than FA grade BSA, suggesting FAF grade BSA has better antifouling properties. Conclusion Herein, we show the presence of BSA reduces the noise within bead-based ELISAs by binding to the bead and blocking nonspecific antibody-bead interactions, regardless of BSA grade, bead type, and pH. FAF grade BSA demonstrates slightly better blocking than FA grade BSA, but both BSA grades decreased nonspecific antibody-bead interactions. Further, BSA shows antifouling properties at the typical assay pH of 7, but lowering the pH to 5.2 can significantly increase BSA bead coating.

Routine MRI Among Patients With a Suspected Scaphoid Fracture Risks Overdiagnosis.

In the setting of a suspected scaphoid fracture, MRI may result in overdiagnosis and potential overtreatment. This is in part because of the low prevalence of true fractures among suspected fractures, but also because of potentially misleading variations in signal that may be more common than fracture-related signal changes. To better understand the risk of overdiagnosis, we first need insight into the relative prevalence of useful and potentially distracting signal changes among patients with a suspected scaphoid fracture. What is the proportion of signal changes representing definite and possible scaphoid fractures relative to other types of signal changes on MRI among patients with a suspected scaphoid fracture? In a retrospective study in an orthopaedic trauma clinic associated with a Level I trauma center, we evaluated MR images of patients 16 years and older with a clinically suspected scaphoid fracture. At our institution, patients with symptoms and signs of a possible scaphoid fracture and negative radiographs undergo MRI scanning. Between January 1, 2012, and September 1, 2019, a total of 310 patients 16 years or older had an MRI to evaluate a suspected scaphoid fracture. Exclusion criteria included a scaphoid fracture that was visible on radiographs before MRI as reported by the radiologist (four patients), no available radiographs before MRI (two), MRI more than 3 weeks after injury (28), unknown date of injury (nine), and repeat or bilateral MRI scans (11), leaving 256 MR images for analysis. Sixty percent (153) of patients were women, and the median age was 34 years (IQR 21 to 50 years). The images were taken a median of 8 days (IQR 2 to 12 days) after injury. MR images were screened for the presence of scaphoid signal changes. We identified the following patterns of signal change with a reliability of kappa 0.62: definite scaphoid fracture, possible scaphoid fracture, signal in the waist area other than possible or definite fractures, and other signal changes. A definite scaphoid fracture was defined as a linear, focal, and bicortical signal abnormality, with adjacent edema and a relatively transverse orientation relative to the scaphoid long axis. The transverse linear signal was visible on more than one cut in multiple planes. A possible scaphoid fracture had a transverse linear signal on more than one cut on sagittal or coronal planes, with or without adjacent edema. Six percent (16 of 256) of MR images were categorized as revealing definite (2% [four of 256]) or possible (5% [12 of 256]) scaphoid fractures, whereas 29% (74 of 256) were categorized as revealing nonspecific signal changes at the waist (14% [35 of 256]) and other areas (15% [39 of 256]). Of the 51 patients with scaphoid waist signal changes, 69% (35) were categorized as having distracting and potentially misleading MRI findings. The high prevalence of signal changes that are distracting and potentially misleading, the low prevalence of signal changes that clearly represent a scaphoid fracture, and the low pretest odds of a true fracture among patients with a suspected scaphoid fracture illustrate that routine MRI of suspected scaphoid fractures carries a notable risk of overdiagnosis and potential overtreatment. Two alternative strategies are supported by preliminary evidence and merit additional attention: more-selective use of MRI in people deemed at higher risk according to a clinical prediction rule and strategies for involving the patient in decisions regarding how to manage the notably small risk of future symptomatic nonunion. Level IV, diagnostic study.

Quantitative Polymerase Chain Reaction System for Alongshan Virus Detection.

The recently discovered Jingmenvirus group includes viruses with a segmented genome, RNA of a positive polarity, and several proteins with distant homology to the proteins of the members of the genus Orthoflavivirus. Some Jingmenvirus group members, namely the Alongshan virus (ALSV) and Jingmen tick virus, are reported to be tick-borne human pathogens that can cause a wide variety of symptoms. The ALSV is widely distributed in Eurasia, yet no reliable assay that can detect it exists. We describe a qPCR system for ALSV detection. Our data showed that this system can detect as little as 104 copies of the ALSV in a sample. The system showed no amplification of the common tick-borne viruses circulating in Eurasia, i.e., the Yanggou tick virus-which is another Jingmenvirus group member-or some known members of the genus Orthoflavivirus. The qPCR system was tested and had no nonspecific signal for the Ixodes ricinus, I. persulcatus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, and H. japonica ticks. The qPCR system had no nonspecific signal for human and sheep serum as well. Overall, the qPCR system described here can be used for reliable and quantitative ALSV detection.

The Clinical Characteristics, Diagnostic Methods, Treatment, and Outcomes of Listeria monocytogenes Meningoencephalitis: A Case Series Study from China.

Listeria monocytogenes meningoencephalitis (LMM) is a rare but dangerous infection of the nervous system. This study intends to summarize and discuss the clinical features, diagnostic methods, treatment, and outcomes. LMM patients treated in the First Affiliated Hospital of Zhengzhou University from June 2013 to December 2022 were retrospectively studied. The clinical data, magnetic resonance imaging (MRI), biochemical and hematological parameters, pathogen tests, treatments, and outcomes information were collected and summarized. Ten LMM patients were enrolled in this study. Three were female and seven were male, with a mean age of 54.5 years old. Seven (70%) of the patients had chronic conditions that either affected the liver (1 case), heart (1 case), or immune system (5 cases). The main clinical manifestations were fever (100%), headache, (60%), and disturbance of consciousness (60%). Out of the nine patients taken MRI, enhanced magnetic resonance imaging was observed with cerebral parenchyma and meningeal enhancement in 3 patients, whereas six patients showed non-specific abnormal signals and brain edema. Seven (70%) patients were positive for L. monocytogenes by bacterial culture and five patients were positive by further next-generation sequencing (NGS) test in CSF. All patients were treated with antibiotics based on antibiotic sensitivity tests or experience, with seven (70%) improved but three (30%) died within one month. LMM patients have a high mortality rate. Considering the time and reliability of NGS reports, it would be better to identify L. monocytogenes infection in the CSF than bacterial culture. While, after diagnosis, giving timely and appropriate antibiotics would still be a challenge to achieve good outcomes.

Utility of Gadolinium-Based Contrast in Initial Evaluation of Seizures in Children Presenting Emergently.

The frequency and utility of gadolinium in evaluation of acute pediatric seizure presentation is not well known. The purpose of this study was to assess the utility of gadolinium-based contrast agents in MR imaging performed for the evaluation of acute pediatric seizure presentation. We identified consecutive pediatric patients with new-onset seizures from October 1, 2016, to September 30, 2021, who presented to the emergency department and/or were admitted to the inpatient unit and had an MR imaging of the brain for the evaluation of seizures. The clinical and imaging data were recorded, including the patient's age and sex, the use of IV gadolinium, and the underlying cause of epilepsy when available. A total of 1884 patients were identified for inclusion. Five hundred twenty-four (28%) patients had potential epileptogenic findings on brain MR imaging, while 1153 (61%) patients had studies with normal findings and 207 (11%) patients had nonspecific signal changes. Epileptogenic findings were subclassified as the following: neurodevelopmental lesions, 142 (27%); intracranial hemorrhage (traumatic or germinal matrix), 89 (17%); ischemic/hypoxic, 62 (12%); hippocampal sclerosis, 44 (8%); neoplastic, 38 (7%); immune/infectious, 20 (4%); phakomatoses, 19 (4%); vascular anomalies, 17 (3%); metabolic, 3 (<1%); and other, 90 (17%). Eight hundred seventy-four (46%) patients received IV gadolinium. Of those, only 48 (5%) cases were retrospectively deemed to have necessitated the use of IV gadolinium: Fifteen of 48 (31%) cases were subclassified as immune/infectious, while 33 (69%) were neoplastic. Of the 1010 patients with an initial noncontrast study, 15 (1.5%) required repeat MR imaging with IV contrast to further evaluate the findings. Gadolinium contrast is of limited additive benefit in the imaging of patients with an acute onset of pediatric seizures in most instances.

Role of Imaging in Diagnosis of Neurological Complications of COVID-19

Background The COVID-19 pandemic has become a challenging world issue after its emergence in December 2019. Despite its most characteristic symptom of respiratory distress, patients with COVID-19 have also shown neurologic manifestations. Aim of the Work To investigate the yield of neuroimaging in patients with COVID-19 undergoing CT or MR imaging of the brain and to describe associated imaging findings. Patients and Methods A descriptive cross–sectional study conducted at Ain Shams University Hospitals over a course of eight months from December 2020 to July 2021. All patients with confirmed COVID-19 infection who presented with neurological symptoms and underwent neuroimaging were included. Results 39 patients were included in the study, with both adults (84.6%) and children (15.4%). Acute ischemic stroke is found to be the most common complication among adult group (48.5%) followed by Post-covid invasive fungal sinusitis (45.5%). Among the children, the neurological complications are rare but severe including vasculitis (50%), infarctions (33.3 %), cerebritis (16.7 %), venous sinus thrombosis (16.7 %), PRES (16.7 %) and non-specific white matter signal abnormality (16.7 %). Conclusion COVID-19 primarily affects the respiratory system. However, neurological involvement is not uncommon and can result in serious complications if not detected and managed early. These complications in some cases can even precede the respiratory symptoms or may be the only symptoms in COVID-19 patients. Therefore, a high index of suspicion is required while dealing with such cases for proper treatment and prevention.

Dual-Signal Cascaded Nucleic Acid Amplification Circuit-Loaded Metal-Organic Frameworks for Accurate and Robust Imaging of Intracellular MicroRNA.

Cascaded signal amplification technologies play an important role in the sensitive detection of lowly expressed biomarkers of interests yet are constrained by severe background interference and low cellular accessibility. Herein, we constructed a metal-organic framework-encapsulating dual-signal cascaded nucleic acid sensor for precise intracellular miRNA imaging. ZIF-8 nanoparticles load and deliver FAM-labeled upstream catalytic hairpin assembly (CHA) and Cy5-modified downstream hybridization chain reaction (HCR) hairpin reactants to tumor cells, enabling visualization of the target-initiated signal amplification process for double-insurance detection of analytes. The pH-responsive ZIF-8 nanoparticles effectively protect DNA hairpins from degradation and allow the release of them in the acid tumor microenvironment. Then, intracellular target miRNAs orderly trigger cascaded nucleic acid signal amplification reaction, of which the exact progress is investigated through the analysis of the fluorescence recovering process of FAM and Cy5. In addition, DNA@ZIF-8 nanoparticles improve measurement accuracy by dual-signal colocalization imaging, effectively avoiding nonspecific false-positive signals and enabling in situ imaging of miRNAs in living cells. A dual-signal colocalization strategy allows accurate target detection in living cells, and DNA@ZIF-8 provides a promising intracellular sensing platform for signal amplification and visual monitoring.

Incubation of protonated NADH or NADPH with ortho-aminobenzaldehyde generates a novel fluorescent nicotinamide dihydroquinazoline condensate

Incubation of reduced nicotinamide adenine dinucleotide (NADH) but not oxidized NAD+ with ortho-aminobenzaldehyde (oABA) generated an uncharacterized chromophore with an absorption peak characteristic of a dihydroquinazoline condensate. This chromophore is responsible for a non-specific signal in a diamine oxidase (DAO) activity assay based on the generation of fluorescent dihydroquinazoline structures directly from DAO substrates.Herein we show that at pH values below 3.0 the glycosidic bond of NADH/NADPH is broken releasing double protonated dihydro-nicotinamide (dihydro-NAM), which consequently condensates with oABA to a novel dihydroquinazoline chromophore and fluorophore, namely the 6- or 8-carbamoyl-5H,7H,8H,9H-10λ⁵-pyrido[2,1-b]quinazolin-10-ylium isomer (CMPQ). The second protonation event closely correlates with the pKa of the N1 nitrogen of C5-protonated dihydro-NAM and fluorophore stability. The fusion partner of oABA is likely the iminium of the primary acid product of dihydro-NAM after glycosidic bond hydrolysis and before irreversible cyclization.Trapping of protonated dihydro-NAM from NADH or NADPH with oABA allows quantification of these dinucleotides. Despite almost a century of research studying acid-catalyzed molecular rearrangements of NADH and NADPH, new and surprising details can be discovered.

A pH-responsive magnetic resonance tuning probe for precise imaging of bacterial infection in vivo.

Accurate and sensitive detection of bacteria is essential for treating bacterial infections. Herein, a pH-responsive magnetic resonance tuning (MRET) probe, whose T1-weighted signal is activated in the bacteria-infected acid microenvironment, is developed for in situ accurately magnetic resonance imaging (MRI) of bacterial infection in vivo. The MRET probe (MDVG-1) is an assembly of paramagnetic enhancer (gadolinium-modified i-motif DNA3, abbreviated as Gd-DNA3-Gd) and the precursor of superparamagnetic quencher (DNA and vancomycin-modified magnetic nanoparticle, abbreviated as MDV). The T1-weighted signal of Gd-DNA3-Gd is quenched once the formation of MDVG-1 (MRET ON). Interestingly, the MDVG-1 probe was disassembled into the monomers of Gd-DNA3-Gd and MDV under the bacteria-infected acid microenvironment, resulting significantly enhanced T1-weighted signal at the infected site (MRET OFF). The pH-responsive MRET probe-based enhanced MRI signal and bacteria targeting significantly improve the distinction between bacterial infectious tissues and sterile inflamed tissues, which provides a promising approach for accurately detecting bacterial infection in vivo. STATEMENT OF SIGNIFICANCE: : Detecting pathogenic bacteria in vivo based on magnetic resonance imaging (MRI) strategy has been exploring recently. Although various bacterial-targeted MRI probes have been developed to image bacteria in vivo, the MRI signal of these MRI probes is always "on", which inevitably generates nonspecific background MRI signals, affecting the accuracy of MRI to a certain extent. In the current study, based on the magnetic resonance tuning (MRET) phenomenon, we present a pH-responsive MRET probe (MDVG-1) with T2-weighted imaging to T1-weighted imaging switchable properties to achieve in situ precise imaging of bacterial infection in vivo.

Aromatic Ring–Mediated Nonspecific Signaling Mechanism and Nafion‐Dominated Solution in Graphene Field‐Effect Transistor–Based Nucleic Acid Biosensors

AbstractGraphene field‐effect transistors (G‐FETs) have attracted widespread attention in disease diagnosis, benefiting from these advantages of high sensitivity, label‐free, easy integration, and direct detection of nucleic acids (NAs) in liquid environments. However, the problem of nonspecific signals in G‐FETs is not fundamentally solved due to a lack of systematic theoretical research to support the development of effective solutions. Thus, researchers have to rely on speculative mechanisms to minimize nonspecific signals in experiments as much as possible. Herein, the nonspecific signal mechanism caused by eight types of π–π interaction paths mediated by aromatic rings is theoretically determined. Based on theoretical simulation results, the feasibility of blocking nonspecific signal paths through Nafion functionalization methods is experimentally verified. Experiments confirm that Nafion‐modified G‐FETs (NMG‐FETs) have excellent performance in avoiding nonspecific signals compared to traditional G‐FETs. Furthermore, the NMG‐FET achieves ultra‐sensitive detection of Down syndrome–related DNA down to 1 aM and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA down to 5 aM, and shows good specificity in base recognition. This study is expected to promote the theoretical advancement of the nonspecific signal mechanism in G‐FET NA detection and offer a practical strategy for improving signal purity and accuracy.

Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent amplification.

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed. It mainly includes a linear reverse transcription step using tailored-designed target specific capture primers, followed by an exponential amplification process using two universal primers to execute the multiplex assay. For proof of concept, four miRNAs were used as models to develop a multiplexed detection assay within one tube simultaneously and then evaluate the performance of the established STEM-Mi-PCR. The sensitivity of the 4-plexed assay was approximately 100 aM with an equivalent amplification efficiency (95.67±8.58%), and had no cross-reactivity each other with high specificity. Quantification of different miRNAs in twenty patients' tissues shown variation from approximately pM to fM concentration level, demonstrating the possibility of practical application of the established method. Moreover, this method was extraordinarily capable of single nucleotide mutation discrimination in different let-7 family members with no more than 0.7% nonspecific detection signal. Hence, the STEM-Mi-PCR we proposed here paves an easy and promising way for miRNA profiling in future clinical applications.

Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data.

Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein-DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a "sensor" for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2. We also tested a DNA-binding mutant approach and showed that, when feasible, ChIP of a site-specific DNA-binding mutant of the target protein is likely an ideal control. These methods vastly improve our ChIP-seq results in S. cerevisiae and should be applicable in other systems.

FDG PET (and MRI) for Monitoring Immunotherapy in Alzheimer Disease.

Passive immunotherapy for Alzheimer disease has been tried for over 10 years without success. However, in 2021 and most recently in January 2023, the US Food and Drug Administration granted accelerated approval of 2 antibodies for this purpose, aducanumab and lecanemab. In both cases, the approval was based on a presumed therapy-related removal of amyloid deposits from the brain and, in the case of lecanemab, also some delay in cognitive decline. We question the validity of the evidence for the removal of amyloid in particular as assessed by amyloid PET imaging, believing that what is observed is more likely a large nonspecific amyloid PET signal in the white matter that diminishes during immunotherapy-in line with dose-dependent increases in amyloid-related imaging abnormalities and increased loss of cerebral volume in treated compared with placebo patients. To investigate this further, we recommend repeat FDG PET and MRI in all future immunotherapy trials.

Development of a novel PET ligand, [11C]GO289 targeting CK2 expressed in the brain

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image β-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to β-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation

ABSTRACT The main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in circulating SARS-CoV-2 variants has raised concerns about potential resistance. Both emphasize the need for a more reliable, sensitive, and facile 3CLpro assay. Here, we report an orthogonal dual reporter-based gain-of-signal assay for measuring 3CLpro activity in living cells. It builds on the finding that 3CLpro induces cytotoxicity and reporter expression suppression, which can be rescued by its inhibitor or mutation. This assay circumvents most limitations in previously reported assays, especially false positives caused by nonspecific compounds and signal interference from test compounds. It is also convenient and robust for high throughput screening of compounds and comparing the drug susceptibilities of mutants. Using this assay, we screened 1789 compounds, including natural products and protease inhibitors, with 45 compounds that have been reported to inhibit SARS-CoV-2 3CLpro among them. Except for the approved drug PF-07321332, only five of these inhibit 3CLpro in our assays: GC376; PF-00835231; S-217622; Boceprevir; and Z-FA-FMK. The susceptibilities of seven 3CLpro mutants prevalent in circulating variants to PF-07321332, S-217622, and GC376 were also assessed. Three mutants were identified as being less susceptible to PF-07321322 (P132H) and S-217622 (G15S, T21I). This assay should greatly facilitate the development of novel 3CLpro-targeted drugs and the monitoring of the susceptibility of emerging SARS-CoV-2 variants to 3CLpro inhibitors.

Thresholding CITE-Seq-based Antibody Expression Data to Improve Cell Type Identification in scRNA-Seq

Abstract scRNA-seq is widely used to assess immune cell heterogeneity by measuring individual cell transcriptomes. Surface markers have been used for decades to type immune cells by flow and more recently mass cytometry. Expression of surface markers is poorly correlated with the mRNA levels of their encoding transcripts. CITE-Seq provides a systematic approach to analyze surface phenotypes along with cell transcriptomes by using oligonucleotide-tagged antibodies. Like flow cytometry, CITE-seq-based assessment of surface markers is complicated by non-specific binding. Here, we introduce and validate a systematic method of thresholding the levels of protein marker expression to address the issue of non-specific antibody binding. To remove the non-specific signal, a threshold value separating noise from expression for each surface marker is obtained by comparing the antibody signal in a cell type known to not express this surface marker. If no negative cell type is available, we use deconvolution of overlapping normal distributions to find the best separator in density plots (Ridgeline plots) by deconvolution (normalmixEM tool) of overlapping normal distributions. In total, 34 of 49 antibodies tested were reliably thresholded, thus effectively removing the noise from the signal. In a validation experiment testing 188 antibodies at 4 different concentrations, 124 were successfully thresholded, including all 34 mentioned above. Supported by the National Institutes of Health (NIH) P01 HL136275

Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Issues with RNF43 antibodies to reliably detect intracellular location.

RNF43 is an important negative regulator of β-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of β-catenin. RNF43 has also been suggested to regulate β-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/β-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers.