Fluorescence Intensity Distribution Research Articles

Studying species distribution in laser-induced plasma by molecular and atomic fluorescence

The spatial distribution profiles of particles in plasma sources push forward our understanding of plasma evolution and physicochemical processes occuring inside. Optical probe methods, such as laser-induced fluorescence, are advanced tools for spatially resolved plasma studies. In our work, we focused on investigation of distribution of neutral Ca and Fe atoms and CaO and FeO molecules in laser-induced plasma by means of laser-induced fluorescence. The development of excitation-emission schemes for Fe and FeO and the practical implementation of schemes for Ca and CaO allowed us to construct distribution maps of these species in laser plasma at 10 and 100 Torr pressures. Both atomic and molecular fluorescence were observed much further from the plasma formation point than the region of bright spontaneous atomic emission. Additionally, by comparing fluorescence intensity distributions with plasma imaging data, we explain the origin of some pecularities in observable plasma inhomogeneity. Distributions of Ca and CaO fluorescence intensity, as well the distribution of CaO/Ca intensity ratio, demostrate that the monoxide is distributed within the plume by the shock wave, but its concentration in the outer layers of plasma is influenced by recombination with atmospheric oxygen.

Aβ oligomers peak in early stages of Alzheimer's disease preceding tau pathology.

Soluble amyloid beta (Aβ) oligomers have been suggested as initiating Aβ related neuropathologic change in Alzheimer's disease (AD) but their quantitative distribution and chronological sequence within the AD continuum remain unclear. A total of 526 participants in early clinical stages of AD and controls from a longitudinal cohort were neurobiologically classified for amyloid and tau pathology applying the AT(N) system. Aβ and tau oligomers in the quantified cerebrospinal fluid (CSF) were measured using surface-based fluorescence intensity distribution analysis (sFIDA) technology. Across groups, highest Aβ oligomer levels were found in A+ with subjective cognitive decline and mild cognitive impairment. Aβ oligomers were significantly higher in A+T- compared to A-T- and A+T+. APOEε4 allele carriers showed significantly higher Aβ oligomer levels. No differences in tau oligomers were detected. The accumulation of Aβ oligomers in the CSF peaks early within the AD continuum, preceding tau pathology. Disease-modifying treatments targeting Aβ oligomers might have the highest therapeutic effect in these disease stages. Using surface-based fluorescence intensity distribution analysis (sFIDA) technology, we quantified Aβ oligomers in cerebrospinal fluid (CSF) samples of the DZNE-Longitudinal Cognitive Impairment and Dementia (DELCODE) cohortAβ oligomers were significantly elevated in mild cognitive impairment (MCI)Amyloid-positive subjects in the subjective cognitive decline (SCD) group increased compared to the amyloid-negative control groupInterestingly, levels of Aβ oligomers decrease at advanced stages of the disease (A+T+), which might be explained by altered clearing mechanisms.

Resistance to CD38 Antigen Targeted Therapy in Multiple Myeloma May Result from Underrecognized Chromosome 4 Gene Deletions

Anti-CD38 monoclonal antibodies (mAbs), daratumumab (dara) and isatuximab (isa), are approved to treat multiple myeloma (MM) in the first line and relapsed/refractory settings. Resistance portends a poor prognosis, yet underlying mechanisms have not been well characterized. Despite generally high CD38 expression on most malignant plasma cells (PCs), antigen density may impact the clinical efficacy of targeted therapy. Unfortunately, therapeutic anti-CD38 mAbs interfere with detection and quantification of CD38 surface level by most conventional clinical flow cytometry assays, limiting characterization of CD38 antigen escape. Tumor cell evasion of antigen specific therapeutics can occur with target loss or downmodulation, a phenomenon conventionally referred to as antigen escape. To elucidate mechanisms of CD38 antigen escape, we evaluated MM patients treated with anti-CD38 mAbs who underwent a marrow evaluation between 1/2017 and 10/2022 at a single institution. Eighty-one of 161 (50%) patients had become clinically refractory over their course. Among 82 (51%) patients with ≥1 chromosomal genomic array test (CGAT) performed, 10 (12%) were found to harbor a CD38 deletion ( Table). With a CGAT coverage of 31%, the estimated prevalence of CD38 deletions was 39%. The median interval from diagnosis to CD38 deletion was calculated as 6.7 years (IQR 2.8 to 11.6) for CD38-deleted patients and estimated as 12.9 years (95% CI 12 to NA; Figure) for all patients. The presence of a CD38 deletion was associated with an increasing number of chromosomal abnormalities on concurrent FISH (OR 2.00, 95% CI 1.33 to 3.29, p=0.002). Specific FISH and flow cytometry findings occurred in conjunction with a CD38 deletion. In 50% of patients, flow cytometry detected a bimodal distribution of CD38 fluorescence intensity, reflecting two populations of malignant plasma cells with distinct CD38 surface levels. No patient showed a uniform malignant plasma cell population with a normal surface CD38 level. When the CD38 (4p15.32) deletion was identified in the context of a large 4p deletion spanning the FGFR3 locus, the FGFR3 (4p16) probe could detect the relevant loss of 4p. This was seen in 6 of 8 patients with a concurrent FGFR3 deletion by CGAT. The proportion of clones harboring a CD38 deletion could be quantified by FISH over time when testing was performed on a CD138-enriched sample. Low-level CD38 deletions (≤25%) did not confer resistance to dara-containing multi-drug regimens. In two cases where such deletions were present at diagnosis, disease control is ongoing in response to dara-Pd and dara-Rd. We suggest that acquired CD38 gene deletions appear to be common, the proportion of patients harboring them increases over time irrespective of prior exposure to CD38 mAbs, and their presence facilitates tumor antigen escape. We demonstrate that CGAT can identify CD38 deletions. Conventional clinical flow cytometry assays are unable to quantify the CD38 surface level due to interference from anti-CD38 mAbs. However, we propose that the finding of a bimodal distribution of CD38 fluorescence by flow cytometry could serve as an initial screen to identify at-risk patients warranting confirmatory CD38 mutational analysis. Following characterization of a deletion, FISH could be used to monitor the clonal level longitudinally. Our suggestion that the presence of a CD38 deletion in >25% of malignant PC clones predicts for reduced CD38 mAb efficacy requires prospective validation.

Disrupted-in-schizophrenia 1 protein aggregates in cerebrospinal fluid are elevated in patients with first-episode psychosis.

The disrupted-in-schizophrenia 1 (DISC1) protein is a key regulator at the intersection of major signaling pathways relevant for adaptive behavior. It is prone to posttranslational changes such as misassembly and aggregation but the significance of such transformations for human mental illness has remained unclear. We aimed to demonstrate the occurrence of DISC1 protein aggregates in patients with first-episode psychosis (FEP). Cerebrospinal fluid samples of patients with FEP (n = 50) and matched healthy controls (HCs; n = 47) were measured by the highly sensitive surface-based fluorescence intensity distribution analysis technology that enables single aggregate detection. We demonstrate that DISC1 protein aggregates are increased in cerebrospinal fluid samples of patients with FEP versus HCs. The concentration was in the low femtomolar range. No correlations were found with specific symptom levels, but the difference was particularly significant in the subset of patients with the diagnoses schizophrenia, unspecified (DSM-IV 295.9) or schizoaffective disorder (DSM-IV 295.70) at 18-month follow-up. DISC1 protein aggregate levels did not significantly change within the 18-month observation interval and were on average higher for individuals carrying the major DISC1 rs821577 allele, before correction. The occurrence of protein aggregates in vivo in patients with psychotic disorders has not been previously reported. It underscores the significance of posttranslational modifications of proteins both as pathogenetic mechanisms and as potential diagnostic markers in these disorders.

Predictive value of cell population data with Sysmex XN-series hematology analyzer for culture-proven bacteremia.

Cell population data (CPD) parameters related to neutrophils, such as fluorescent light intensity (NE-SFL) and fluorescent light distribution width index (NE-WY), have emerged as potential biomarkers for sepsis. However, the diagnostic implication in acute bacterial infection remains unclear. This study assessed the diagnostic value of NE-WY and NE-SFL for bacteremia in patients with acute bacterial infections, and those associations with other sepsis biomarkers. Patients with acute bacterial infections were enrolled in this prospective observational cohort study. For all patients, a blood sample, with at least two sets of blood cultures, were collected at the onset of infection. Microbiological evaluation included examination of the blood bacterial load using PCR. CPD was assessed using Automated Hematology analyzer Sysmex series XN-2000. Serum levels of procalcitonin (PCT), interleukin-6 (IL-6), presepsin, and CRP were also assessed. Of 93 patients with acute bacterial infection, 24 developed culture-proven bacteremia and 69 did not. NE-SFL and NE-WY were significantly higher in patients with bacteremia than in those without bacteremia (p < 0.005, respectively), and were significantly correlated with the bacterial load determined by PCR (r = 0.384 and r = 0.374, p < 0.005, respectively). To assess the diagnostic value for bacteremia, receiver operating characteristic curve analysis was used. NE-SFL and NE-WY showed an area under the curve of 0.685 and 0.708, respectively, while those of PCT, IL-6, presepsin, and CRP were 0.744, 0.778, 0.685, and 0.528, respectively. Correlation analysis showed that the levels of NE-WY and NE-SFL were strongly correlated with PCT and IL-6 levels. This study demonstrated that NE-WY and NE-SFL could predict bacteremia in a manner that may be different from that of other indicators. These findings suggest there are potential benefits of NE-WY/NE-SFL in predicting severe bacterial infections.

Development and Implementation of an Internal Quality Control Sample to Standardize Oligomer-Based Diagnostics of Alzheimer's Disease.

Protein misfolding and aggregation are pathological hallmarks of various neurodegenerative diseases. In Alzheimer's disease (AD), soluble and toxic amyloid-β (Aβ) oligomers are biomarker candidates for diagnostics and drug development. However, accurate quantification of Aβ oligomers in bodily fluids is challenging because extreme sensitivity and specificity are required. We previously introduced surface-based fluorescence intensity distribution analysis (sFIDA) with single-particle sensitivity. In this report, a preparation protocol for a synthetic Aβ oligomer sample was developed. This sample was used for internal quality control (IQC) to improve standardization, quality assurance, and routine application of oligomer-based diagnostic methods. We established an aggregation protocol for Aβ1-42, characterized the oligomers by atomic force microscopy (AFM), and assessed their application in sFIDA. Globular-shaped oligomers with a median size of 2.67 nm were detected by AFM, and sFIDA analysis of the Aβ1-42 oligomers yielded a femtomolar detection limit with high assay selectivity and dilution linearity over 5 log units. Lastly, we implemented a Shewhart chart for monitoring IQC performance over time, which is another important step toward quality assurance of oligomer-based diagnostic methods.

Multicolor flow cytometry in clinical samples for platelet signaling assessment

BackgroundAvailability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. ObjectivesTo describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). MethodsBy comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI–related disease with granule secretion defect). ResultsThe range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. ConclusionIn complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.

Variability of fluorescence intensity distribution measured by flow cytometry is influenced by cell size and cell cycle progression

The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.

Patients with isolated REM-sleep behavior disorder have elevated levels of alpha-synuclein aggregates in stool

Misfolded and aggregated α-synuclein is a neuropathological hallmark of Parkinson’s disease (PD). Thus, α-synuclein aggregates are regarded as a biomarker for the development of diagnostic assays. Quantification of α-synuclein aggregates in body fluids is challenging, and requires highly sensitive and specific assays. Recent studies suggest that α-synuclein aggregates may be shed into stool. We used surface-based fluorescence intensity distribution analysis (sFIDA) to detect and quantify single particles of α-synuclein aggregates in stool of 94 PD patients, 72 isolated rapid eye movement sleep behavior disorder (iRBD) patients, and 51 healthy controls. We measured significantly elevated concentrations of α-synuclein aggregates in stool of iRBD patients versus those of controls (p = 0.024) or PD patients (p < 0.001). Our results show that α-synuclein aggregates are excreted in stool and can be measured using the sFIDA assay, which could support the diagnosis of prodromal synucleinopathies.

Ultrawide Range of Concentration Measurement Assisted by Fluorescence Intensity Distribution Imaging

Ultrawide Range of Concentration Measurement Assisted by Fluorescence Intensity Distribution Imaging

Local pH mapping in the cell adhesion nano-interfaces on a pH-responsive fluorescence-dye-immobilized substrate.

Cell-substrate adhesion nano-interfaces can, in principle, exhibit a spatial distribution of local pH values under the influence of the weakly acidic microenvironment of glycocalyx grafted on lipid bilayer cell membrane which is compressed and closely attached to culture substrate in the vicinity of integrin-adhesion complexes. However, a simple local pH distribution imaging methodology has not been developed. In this study, to visualize the local pH distribution at the cell adhesion interface, we prepared glass substrates chemically modified with a pH-responsive fluorescent dye fluorescein isothiocyanate (FITC), observed the distribution of FITC fluorescence intensity at the adhesion interface of fibroblast (NIH/3T3) and cancer cells (HeLa), and compared the FITC images with the observed distribution of focal adhesions. FITC images were converted to pH mapping based on the pH-fluorescence calibration data of surface-immobilized FITC pre-measured in different pH media, which showed significantly larger regions with lowered pH level (6.8-7.0) from outside the cell (pH 7.4) were observed at the thick inner periphery of HeLa cells while 3T3 cells exhibited smaller lowered pH regions at the thin periphery. The lowered pH regions overlapped with many focal adhesions, and image analysis showed that larger focal adhesions tend to possess more lowered pH sites inside, reflecting enhanced glycocalyx compression due to accumulated integrin-adhesion ligand binding. This tendency was stronger for HeLa than for 3T3 cells. The role of glycocalyx compression and the pH reduction at the cell adhesive interface is discussed.

Radio-fluorogenic nanoclay gel dosimeters with reduced linear energy transfer dependence for carbon-ion beam radiotherapy.

The precise assessment of the dose distribution of high linear energy transfer (LET) radiation remains a challenge, because the signal of most dosimeters will be saturated due to the high ionization density. Such measurements are particularly important for heavy-ion beam cancer therapy. On this basis, the present work examined the high LET effect associated with three-dimensional gel dosimetry based on radiation-induced chemical reactions. The purpose of this study was to create an ion beam radio-fluorogenic gel dosimeter with a reduced effect of LET. Nanoclay radio-fluorogenic gel (NC-RFG) dosimeters were prepared, typically containing 100μM dihydrorhodamine 123 (DHR123) and 2.0wt% nanoclay together with catalytic additives promoting Fenton or Fenton-like reactions. The radiological properties of NC-RFG dosimeters having different compositions in response to a carbon-ion beam were investigated using a fluorescence gel scanner. An NC-RFG dosimeter capable of generating a fluorescence intensity distribution reflecting the carbon-ion beam dose profile was obtained. It was clarified that the reduction of the unfavorable LET dependence results from an acceleration of the reactions between DHR123 and H2 O2 , which is a molecular radiolysis product. The effects of varying the preparation conditions on the radiological properties of these gels were also examined. The optimum H2 O2 catalyst was determined to include 1mM Fe3+ ions, and the addition of 100mM pyridine was also found to increase the sensitivity. This technique allows the first-ever evaluation of the depth-dose profile of a carbon-ion beam at typical therapeutic levels of several Gy without LET effect.

An ER-targeted “reserve-release” fluorogen for topological quantification of reticulophagy

The endoplasmic reticulum's (ER) dynamic nature, essential for maintaining cellular homeostasis, can be influenced by stress-induced damage, which can be assessed by examining the morphology of ER dynamics and, more locally, ER properties such as hydrophobicity, viscosity, and polarity. Although numerous ER-specific chemical probes have been developed to monitor the ER's physical and chemical parameters, the quantitative detection and super-resolution imaging of its local hydrophobicity have yet to be explored. Here, we describe a photostable ER-targeted probe with high signal-to-noise ratio for super-resolution imaging that can specifically respond to changes in ER hydrophobicity under stress based on a “reserve-release” mechanism. The probe shows an excellent ability to target ER over commercial ER dyes and can be used to track local changes of hydrophobicity by fluorescence intensity and morphology during the selective autophagy of ER (i.e., reticulophagy). By correlating the level and location of ER damage with the distribution of fluorescence intensity, we were able to assess reticulophagy at the subcellular level. Beyond that, we developed a topological analytical tool adaptable to any ER probe for detecting structural changes in ER and thus quantitatively identifying reticulophagy. The algorithm-assisted tool can also be adapted to a wide range of molecular probes and organelles. Altogether, the new probe and analytical strategy described here show promise for the quantitative detection and analysis of subtle ER damage and stress.

Analysis of In Vivo Radachlorin Accumulation through FLIM-Assisted Examination of Ex Vivo Histological Samples

We report an investigation of the in vivo accumulation of Radachlorin photosensitizer in a murine model in several types of normal and tumor tissues based on an FLIM-assisted analysis of fluorescence intensity images, time-resolved fluorescence signals, and phasor plots. Experiments were performed on ex vivo histological samples of normal and tumor tissues. It was shown that the investigation of fluorescence intensity distributions combined with that of time-resolved fluorescence images can be used for qualitative and—under some limitations—quantitative analyses of the relative uptake of this photosensitizer in tissues. The phasor plot representations of time-resolved fluorescence signals were shown to be suitable for identification of the accumulation of predominant photosensitizers in tissues.

Co-expression of an isopropanol synthetic operon and eGFP to monitor the robustness of Cupriavidus necator during isopropanol production

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered.

One-Step Digital Droplet Auto-Catalytic Nucleic Acid Amplification with High-Throughput Fluorescence Imaging and Droplet Tracking Computation.

Digital droplet technology has emerged as a powerful new tool for biomarker analysis. Temperature cycling, enzymes, and off-chip processes are, nevertheless, always required. Herein, we constructed a digital droplet auto-catalytic hairpin assembly (ddaCHA) microfluidic system to achieve digital quantification of single-molecule microRNA (miRNA). The designed continuous chip integrates droplet generation, incubation, and fluorescence imaging on the chip, avoiding the requirement for extra droplet re-collection and heating operations. Clearly, the digital readout was obtained by partitioning miRNA into many individual pL-sized small droplets in which the target molecule is either present ("positive") or absent ("negative"). Importantly, the suggested enzyme-free auto-catalytic hairpin assembly (aCHA) in droplets successfully mitigated the effects of the external environment and thermal cycling on droplets, and its reaction rate is significantly superior to that of traditional CHA. We got excellent sensitivity with a linear correlation from 1 pM to 10 nM and a detection limit of 0.34 pM in the fluorescence spectrum section, as well as high selectivity to other miRNAs. Furthermore, the minimum target concentration could be reduced to 10 fM based on the high-throughput tracking computation of fluorescent droplets with a self-developed Python script, and the fluorescence intensity distribution agreed well with the theoretical value, demonstrating that it is feasible to detect miRNA efficiently and accurately, which has great potential applications in clinical diagnostics and biochemical research.

Monitoring DNA polymerase β mitochondrial localization and dynamics

Mouse fibroblasts lacking (null) DNA polymerase β (pol β) were transfected with fluorescently tagged pol β and stained with biomarkers to allow visualization within living cells by confocal microscopy. Transient transfection resulted in varying pol β expression levels. Separating cells into three groups based on pol β fluorescence intensity and morphological distribution, permitted analysis of the concentration dependence and spatial distribution of cytoplasmic pol β. Colocalization between pol β and mitochondria was pol β concentration dependent. A decrease in overlap with nucleoids containing mitochondrial DNA (mtDNA) was observed at the highest pol β intensity where pol β exhibits a tubular appearance, suggesting the ability to load elevated levels of pol β into mitochondria readily available for relocation to damaged mtDNA. The dynamics of pol β and mitochondrial nucleoids were followed by confocal recording of time series images. Two populations of mitochondrial nucleoids were observed, with and without pol β. Micro-irradiation, known to form DNA single-strand breaks, in a line across nucleus and cytoplasm of pol β stably transfected cells enhanced apparent localization of pol β with mitochondria in the perinuclear region of the cytoplasm near the nuclear membrane. Exposure of pol β expressing cells to H2O2 resulted in a time-dependent increase in cytoplasmic pol β observed by immunofluorescence analysis of fixed cells. Further screening revealed increased levels of colocalization of pol β with a mitochondrial probe and an increase in oxidative DNA damage in the cytoplasm. ELISA quantification confirmed an increase of an oxidative mitochondrial base lesion, 7,8-dihydro-8-oxoguanine, after H2O2 treatment. Taken together, the results suggest that pol β is recruited to mitochondria in response to oxidatively-induced mtDNA damage to participate in mtDNA repair.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson’s disease (PD), dementia with Lewy bodies (DLB), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

Sensitively detecting antigen of SARS-CoV-2 by NIR-II fluorescent nanoparticles.

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an efficient way to prevent the spread of coronavirus disease 2019 (COVID-19). Detecting SARS-CoV-2 antigen can be rapid and convenient, but it is still challenging to develop highly sensitive methods for effective diagnosis. Herein, a lateral flow assay (LFA) based on fluorescent nanoparticles emitting in the second near-infrared (NIR-II) window is developed for sensitive detection of SARS-CoV-2 antigen. Benefiting from the NIR-II fluorescence with high penetration and low autofluorescence, such NIR-II based LFA allows enhanced signal-to-background ratio, and the limit of detection is down to 0.01 ng·mL−1 of SARS-CoV-2 antigen. In the clinical swab sample tests, the NIR-II LFA outperforms the colloidal gold LFA with higher overall percent agreement with the polymerase chain reaction test. The clinical samples with low antigen concentrations (∼ 0.015−∼ 0.068 ng·mL−1) can be successfully detected by the NIR-II LFA, but fail for the colloidal gold LFA. The NIR-II LFA can provide a promising platform for highly sensitive, rapid, and cost-effective method for early diagnosis and mass screening of SARS-CoV-2 infection.Electronic Supplementary MaterialSupplementary material (the operation procedure and cost of the materials needed of NIR-II lateral flow assays, the dynamic light scattering spectrum of the NIR-II nanoparticles, the components and testing principle, optimization of main parameters pertaining to the LFA performance, the colloidal gold LFA strip, the fluorescence intensity distribution curves and the T/C values of the strips for clinical samples by NIR-II LFA, and results of clinical swab samples detected by colloidal gold LFA) is available in the online version of this article at 10.1007/s12274-022-4351-1.

Aβ oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo

SummaryThe elimination of amyloid beta (Aβ) oligomers is a promising strategy for therapeutic drug development of Alzheimer’s disease (AD). AD mouse models that develop Aβ pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aβ oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aβ oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.