Direct Readout Research Articles

A molecular imprint of intracellular amyloid-beta ApoE interaction correlates with its toxicity molecular imprint of intracellular amyloid beta ApoE interaction.

ApoE isoforms are major genetic risk factors related to Alzheimer's disease (AD), which is associated with the aggregation of the amyloid beta (Aβ) peptide. However, a molecular-level understanding of how ApoE is involved in AD pathology is still lacking. Here we employ a fluorescent lifetime-based assay that reports a specific ApoE-dependent modification of Aβ, both in vitro and in neuronal cells. We explore this phenomenon in several types of cells where the change of lifetime of Aβ oligomers correlates with the relative ApoE content, with the highest effect displayed by primary-cultured rat astrocytes. At the single-molecule level, ApoE-induced modification also produces distinct Aβ oligomers with increased affinity to artificial lipid bilayers. A known peptide inhibitor of Aβ-ApoE interaction, LVFFA, diminishes both the lifetime modification and toxicity of Aβ oligomers in a dose-dependent manner. The correlation between the short lifetime component and Aβ toxicity was corroborated by experiments using two nontoxic Aβ variants. Our data, therefore, establish a clear correlation between the lifetime modification of Aβ and its toxicity. Finally, neuronal stem cells derived from cells obtained from Alzheimer's patients also show the short-lifetime component, the changed oligomeric state, and the increased membrane affinity, which is likely to be relevant for AD. In summary, our assay can provide a direct readout of the toxic ApoE-Aβ interaction in vitro and in cells, which can provide a platform for AD drug discovery.

A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency.

Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.

The ambivalence toward neuropsychology in dementia research, diagnosis, and drug development: Myths and misconceptions.

Clinical assessment remains the gold standard for diagnosing dementia, monitoring progression, and conducting clinical research. Biomarkers hold promise for targeted therapeutic approaches, selection of participants in clinical trials, and direct physiological efficacy readouts. However, the anchoring of biomarker research to clinical symptomatology is often based on short and insensitive cognitive screening. This gives the impression that cognitive symptoms occur relatively late and that their progression in the early stages of the disease is slow. A thorough cognitive assessment is a powerful tool and has a key role in the accurate and early diagnosis of dementia. It is very different from the cognitive testing usually seen in biomarker research and drug development. Yet the distinction between these approaches is unclear to many. This paper highlights the misconceptions around cognitive research in dementia and suggests a way forward to facilitate biomarker and drug development through the improved utility of cognitive assessment tools.

Exploiting ionization dynamics in the nitrogen vacancy center for rapid, high-contrast spin, and charge state initialization

We propose and experimentally demonstrate a method to strongly increase the sensitivity of spin measurements on nitrogen vacancy (NV) centers in diamond, which can be readily implemented in existing quantum sensing experiments. While charge state transitions of this defect are generally considered a parasitic effect to be avoided, we show here that these can be used to significantly increase the NV center's spin contrast, a key quantity for high-sensitivity magnetometry and high-fidelity state readout. The protocol consists of a two-step procedure, in which the charge state of the defect is first purified by a strong laser pulse, followed by weak illumination to obtain high spin polarization. We observe a relative improvement of the readout contrast by $17%$ and infer a reduction of the initialization error of more than 50%. The contrast enhancement is accompanied by a beneficial increase of the readout signal. For long sequence durations, typically encountered in high-resolution magnetometry, a measurement speedup by a factor of $>1.5$ is extracted, and we find that the technique is beneficial for sequences of any duration. Additionally, our findings give detailed insight into the charge and spin polarization dynamics of the NV center and provide actionable insights for direct optical, spin-to-charge, and electrical readout of solid-state spin centers.

A Telomerase-Activated Magnetic Resonance Imaging Probe for Consecutively Monitoring Tumor Growth Kinetics and In Situ Screening Inhibitors.

Telomerase has long been considered as a biomarker for cancer diagnosis and a therapeutic target for drug discovery. Detecting telomerase activity in vivo could provide more direct information of tumor progression and response to drug treatment, which, however, is hampered by the lack of an effective probe that can generate an output signal without a tissue penetration depth limit. In this study, using the principle of distance-dependent magnetic resonance tuning, we constructed a telomerase-activated magnetic resonance imaging probe (TAMP) by connecting superparamagnetic ferroferric oxide nanoparticles (SPFONs) and paramagnetic Gd-DOTA (Gd(III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complexes via telomerase-responsive DNA motifs. Upon telomerase-catalyzed extension of the primer in TAMP, Gd-DOTA-conjugated oligonucleotides can be liberated from the surface of SPFONs through a DNA strand displacement reaction, restoring the T1 signal of the Gd-DOTA for a direct readout of the telomerase activity. Here we show that, by tracking telomerase activity, this probe provides consistent monitoring of tumor growth kinetics during progression and in response to drug treatment and enables in situ screening of telomerase inhibitors in whole-animal models. This study provides an alternative toolkit for cancer diagnosis, treatment response assessment, and anticancer drug screening.

Biodistribution of Adeno-Associated Virus Gene Therapy Following Cerebrospinal Fluid-Directed Administration.

Adeno-associated virus (AAV)-based gene therapies, exemplified by the approved therapy for spinal muscular atrophy, have the potential to deliver disease-course-altering treatments for central nervous system (CNS) indications. However, several clinical trials have reported severe adverse events, including patient deaths following high-dose systemic administration for muscle-directed gene transfer, highlighting the need to explore approaches utilizing lower doses when targeting the CNS. Animal models of disease provide insight into the response to new AAV therapies. However, translation from small to larger animals and eventually to humans is hampered by anatomical and biological differences across the species and their impact on AAV delivery. We performed a literature review of preclinical studies of AAV gene therapy biodistribution following cerebrospinal fluid (CSF) delivery (intracerebroventricular, intra-cisterna magna, and intrathecal lumbar). The reviewed literature varies greatly in the reported biodistribution of AAV following administration into the CSF. Differences between studies, including animal model, vector serotype used, method used to assess biodistribution, and route of administration, among other variables, contribute to differing outcomes and difficulties in translating these preclinical results. For example, only half of the published AAV-based gene therapy studies report vector copy number, the most direct readout following administration of a vector; none of these studies reported details such as the empty:full capsid ratio and quality of encapsidated genome. Analysis of the last decade's literature focusing on AAV-based gene therapies targeting the CNS underscores limitations of the body of knowledge and room for continued research. In particular, there is a need to understand the biodistribution achieved by different CSF-directed routes of administration and determining if specific cell types/structures of interest will be transduced. Our findings point to a clear need for a more systematic approach across the field to align the assessments and elements reported in preclinical research to enable more reliable translation across animal models and into human studies.

Long-Term Durability and Cycling of Nanoporous Materials Based Impedance NO2 Sensors

Nanoporous materials, including metal–organic frameworks (MOFs) and inorganic zeolites, are gaining attention as gas sensor materials due to their chemical selectivity and robustness. To advance industrial viability of these materials as sensors, long-term, variable environment testing is needed to evaluate their stability and continued chemical exposure response. Nanoporous materials-based direct electrical readout sensors were evaluated for 3 months under dry or humid conditions at 74 °C. The sensors were comprised of either Ni-MOF-74, Mg-MOF-74, or Ni-SSZ-13 zeolite. Additionally, we describe the development of multichambered sensor testing platforms that allows uninterrupted direct impedance monitoring of each sensor over long test periods. Results indicate relative stability in dry conditions for the sensors over time. In contrast, degradation of the active sensing material is evident in the humid environment. Collectively, these results demonstrate need for long-term testing of emerging nanoporous sensor materials under specific environmental conditions.

Edible cellulose-based colorimetric timer.

A biocompatible and edible colorimetric timer is obtained by exploiting the dynamic colour changes of the cholesteric liquid crystalline mesophases of hydroxypropyl cellulose (HPC) in aqueous suspensions. The edible timer is encapsulated between semi permeable membranes made of shellac. The cholesteric organisation of the HPC provides vibrant colouration, while the shellac layers allow tuning of the evaporation rate of water from the mesophase, which results in a colour change. Due to the biocompatibility of the components and the direct read-out of the system, i.e. the colour change can be visually detected, and the developed timer can be implemented as a colorimetric sensor with potential to be used in food packaging, and as a smart labelling system.

Fabrication of CdS quantum dots with egg white and application in the assay of hypochlorous acid and myeloperoxidase activity and inhibition.

The myeloperoxidase (MPO)/H2O2-Cl- enzymatic reaction system and its product hypochlorous acid (HOCl) are closely related to many disease processes, and new methods to detect the levels of HOCl and MPO are being focused on. MPO is the only known enzyme for the catalytic production of HOCl in biological systems; therefore, monitoring the HOCl levels is a selective and direct readout of MPO activity. This study reported a simple and efficient fluorescence assay of HOCl and MPO activity and inhibition. Highly fluorescent CdS quantum dots (CdS QDs) were prepared in one pot where NaOH-pretreated egg white served as a stabilizer. These CdS QDs exhibit strong green emission centered at ca. 550 nm and enable rapid and selective fluorescence response to HOCl with a linear detection range of 8.0-250 μM and a limit of detection (LOD) of 2.5 μM. Moreover, the CdS QDs were further applied for sensing MPO based on the fluorescence quenching exerted by its reaction product HOCl. Detection of MPO is accomplished with a linear range from 0.1 to 40 mU mL-1 (1 U is the MPO concentration for catalysis of 1 micromolar substrate per minute) and a LOD of 0.06 mU mL-1. The developed synthesis method can be applied to large-scale synthesis of CdS QDs, and the strategy to sense HOCl and MPO activity and inhibition has potential biomedical applications such as clinical diagnosis and drug screening.

Coupled folding-upon-binding of human tumor suppressor MIG6 to lung cancer EGFR kinase domain and molecular trimming/stapling of MIG6-derived β-hairpins to target the coupling event.

Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured β-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed β-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.

Thermal Gradient Induced Transparency and Absorption in a Microcavity

AbstractGeneration of electromagnetically induced transparency (EIT) and absorption (EIA) effects in optical cavity systems paves a way for many applications ranging from fundamental physics to various photonic applications, such as analogous quantum interference effects, slow light, and light storage. Here, thermal gradient induced transparency (TGIT) and absorption (TGIA) effects in a single microcavity are proposed and demonstrated. The effects originate from the unique temperature gradient in the axial direction of the functionalized microbottle cavity, which leads to different resonance evolutions for whispering gallery modes with different axial orders. Furthermore, this platform is leveraged as a probe to realize a direct temperature readout from the transmission spectrum via multiple TGIT (TGIA is also applicable)‐based photonic barcodes with a high detection resolution of 9 × 10−4 °C. This platform offers a novel, highly efficient, and simple scheme to realize dynamic mode coupling for various applications, such as light storage and temperature detection.

Arbitrary Polarization Readout with Dual-Channel Neuro-Metasurfaces.

Polarization, as a vector nature of the electromagnetic wave, plays a fundamental role in optics. Determining the polarization state of light is required by many applications, spanning from remote sensing and material analysis to biology and microscopy. To achieve this goal, conventional methods necessitate cascading of multiple optical components and consequential measurements to estimate the Stokes parameters, rendering the entire optical system bulky, complex, and sensitive. Here a brand-new strategy is introduced for direct polarization readout based on dual-channel neuro-metasurfaces. Neuro-metasurfaces can independently manipulate two orthogonal linearly-polarized waves that can synthesize arbitrary polarization waves with a linear combination. By judiciously designing the output focus points, a unique polarization atlas is created that allows one-to-one correspondence from intensity ratio to polarization state. To implement this, polarization-sensitive metasurfaces are designed and the spatial layout is optimized using a diffractive neural network. The feasibility of this strategy is validated by numerical simulation and microwave experiments. These results pave a new avenue in realizing integrated and multifunctional detectors and demonstrate the potential of neuro-metasurfaces as an add-on for discomposing and composing spatial basis.

Impaired Pain Processing at a Brainstem Level Is Involved in Maladaptive Neuroplasticity in Patients with Chronic Complex Regional Pain Syndrome

Neuroinflammatory mechanisms and maladaptive neuroplasticity underlie the progression of complex regional pain syndrome (CRPS), which is prototypical of central neuropathic pain conditions. While cortical maladaptive alterations are well described, little is known about the contribution of the brainstem to the pathophysiology. This study investigates the role of pain-modulatory brainstem pathways in CRPS using the nociceptive blink reflex (nBR), which not only provides a direct read-out of brainstem excitability and habituation to painful stimuli but may also be suitable for use as a diagnostic biomarker for CRPS. Thirteen patients with CRPS and thirteen healthy controls (HCs) participated in this prospective case-control study investigating the polysynaptic trigemino-cervical (R2) nBR response. The R2 area and its habituation were assessed following repeated supraorbital electrical stimulation. Between-group comparisons included evaluations of diagnostic characteristics as a potential biomarker for the disease. Patients with CRPS showed a substantial decrease in habituation on the stimulated (Cohen's d: 1.3; p = 0.012) and the non-stimulated side (Cohen's d: 1.1; p = 0.04). This is the first study to reveal altered nBR habituation as a pathophysiological mechanism and potential diagnostic biomarker in CRPS. We confirmed previous findings of altered nBR excitability, but the diagnostic accuracy was inferior. Future studies should investigate the nBR as a marker of progression to central mechanisms in CRPS and as a biomarker to predict treatment response or prognosis.

Single Cell Gene Expression Predicts Plasma Cell Leukemia Patient Response to Daratumumab

The promise of precision medicine is to identify the ideal treatment for each patient, eliminating failed treatment cycles and reducing treatment burdens on patients and payors. While gene sequencing can predict effective treatment options in some cases, studies have estimated that only 10-15% of cancer patients are treated with genotype matched drugs. Gene expression is theoretically an attractive alternative to predicting therapeutic response because, it provides a direct readout of dysregulated cellular activities including druggable pathways. However, the use of bulk gene expression to predict therapeutic response is hampered by tumor heterogeneity; that is, a biopsied tumor contains multiple cells types and different tumor clones and a bulk analysis only generates an average gene expression signal across all of the cells, leading to a decreased sensitivity for identifying biomarkers found only in specific cell subgroups. More recently, single-cell gene expression analysis has shown promise in improving precision medicine through a focused look into the biology of the tumor cells themselves. For example, we previously showed (Scolnick, et al ASCO 2022) that a model trained on single-cell gene expression data collected from 17 Multiple Myeloma (MM) patient's plasma cells (PCs) could be used to predict the therapeutic response of seven independent MM patient samples at 100% accuracy. Here we combine single-cell gene expression analysis with Singleron Biotechnology's MapResponse™ machine learning algorithms to extend our predictions to data from peripheral blood mononuclear cells (PBMCs) of plasma cell leukemia patients who were treated with daratumumab. Our model accurately predicted the response to daratumumab treatment for both patients based on the PCs collected from blood samples. These early findings point to the potential value of combining single-cell RNA sequencing with machine learning methods such as MapResponse™ to bring precision medicine to more patients based on minimal invasive assessment of PBMCs.

Discovery of pathway-independent protein signatures associated with clinical outcome in human cancer cohorts

Proteomic data provide a direct readout of protein function, thus constituting an information-rich resource for prognostic and predictive modeling. However, protein array data may not fully capture pathway activity due to the limited number of molecules and incomplete pathway coverage compared to other high-throughput technologies. For the present study, our aim was to improve clinical outcome prediction compared to published pathway-dependent prognostic signatures for The Cancer Genome Atlas (TCGA) cohorts using the least absolute shrinkage and selection operator (LASSO). RPPA data is particularly well-suited to the LASSO due to the relatively low number of predictors compared to larger genomic data matrices. Our approach selected predictors regardless of their pathway membership and optimally combined their RPPA measurements into a weighted risk score. Performance was assessed and compared to that of the published signatures using two unbiased approaches: 1) 10 iterations of threefold cross-validation for unbiased estimation of hazard ratio and difference in 5-year survival (by Kaplan–Meier method) between predictor-defined high and low risk groups; and 2) a permutation test to evaluate the statistical significance of the cross-validated log-rank statistic. Here, we demonstrate strong stratification of 445 renal clear cell carcinoma tumors from The Cancer Genome Atlas (TCGA) into high and low risk groups using LASSO regression on RPPA data. Median cross-validated difference in 5-year overall survival was 32.8%, compared to 25.2% using a published receptor tyrosine kinase (RTK) prognostic signature (median hazard ratios of 3.3 and 2.4, respectively). Applicability and performance of our approach was demonstrated in three additional TCGA cohorts: ovarian serous cystadenocarcinoma (OVCA), sarcoma (SARC), and cutaneous melanoma (SKCM). The data-driven LASSO-based approach is versatile and well-suited for discovery of new protein/disease associations.

In-depth Retrospective Review of Originally Negative Screening Mammograms from Women with Confirmed Breast Cancer.

We aim to contribute to the assessment of the screening performance in Flanders (Belgium) and to identify valuable mammograms for subsequent studies and training. Initially negative prior screening mammograms (sMx) of 210 women with confirmed breast cancer detected by the Flemish screening programme between 2011-2013 were reviewed by a highly experienced radiologist. The review of the prior sMx was performed in three steps: 1) only prior mammograms available; 2) with index sMx (=subsequent positive sMx) present; 3) with index sMx and clinical information present. The radiological review yielded 94 (45%) mammograms 'without suspicious lesions', 77 (37%) 'with minimal signs in at least one breast', and 39 (19%) 'with clearly visible tumours'. In univariate analyses, the reclassification of prior sMx was significantly associated with the date of the prior sMx, the need for a third reader for arbitration, image quality and the detector system used (computed radiography versus direct readout digital radiography), and it was not associated with the interval between screening rounds, age at prior sMx, breast density, or tumour characteristics (<T2 versus ≥T2, in situ versus invasive). In multivariate analyses, the date of the prior sMx (p = 0.001), need for arbitration (p = 0.001) and image quality (p < 0.001) remained significantly associated with the reclassification. This retrospective review reclassified 19% of the sMx as clearly visible tumours. With this, the Flemish screening programme performs in accordance with similar studies. The sMx reviewed in this study, form a valuable set of mammograms for training and further research.

Failing to attend versus failing to stop: Single-trial decomposition of action-stopping in the stop signal task

The capacity to stop impending or ongoing actions contributes to executive control over behavior. Action-stopping, however, is difficult to directly quantify. It is therefore assayed via computational modeling of behavior in the stop signal task to estimate the latency of stopping (stop signal reaction time, SSRT) and, more recently, the reliability of stopping in terms of the distribution of SSRTs (standard deviation, SD-SSRT) and the frequency with which one outright fails to react to a stop signal (trigger failures, TF). Critically, the validity of computational estimates remains unknown because we currently have no direct readouts of behavior against which to compare them. Here, we developed a method for providing single-trial behavioral readouts of SSRT and trigger failures. The method relies on an adaptation of the stop signal task in which participants respond by moving a computer mouse. In two online experiments, we used movement kinematics to quantify stopping performance (SSRT, SD-SSRT, and TF), and then applied the standard Race Model and recent BEESTS model in order to examine the convergent validity of the methods. Overall, we demonstrate good correspondence between kinematics- and model-based estimates of stopping performance at the group and individual level. We conclude that the new method provides valid estimates of stopping performance that, unlike model-based estimates, can be read out at the level of single trials. Our approach might therefore be useful for interrogating single-trial neurophysiological correlates of stopping and for large-scale, online studies of behavioral stopping.

Research progress in protein microarrays: Focussing on cancer research.

Although several effective treatment modalities have been developed for cancers, the morbidity and mortality associated with cancer continues to increase every year. As one of the most exciting emerging technologies, protein microarrays represent a powerful tool in the field of cancer research because of their advantages such as high throughput, small sample usage, more flexibility, high sensitivity and direct readout of results. In this review, we focus on the research progress in four types of protein microarrays (proteome microarray, antibody microarray, lectin microarray and reversed protein array) with emphasis on their application in cancer research. Finally, we discuss the current challenges faced by protein microarrays and directions for future developments. We firmly believe that this novel systems biology research tool holds immense potential in cancer research and will become an irreplaceable tool in this field.

Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors.

Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.

Spin and Orbital Spectroscopy in the Absence of Coulomb Blockade in Lead Telluride Nanowire Quantum Dots

We investigate quantum dots in semiconductor PbTe nanowire devices. Due to the accessibility of ambipolar transport in PbTe, quantum dots can be occupied both with electrons and holes. Owing to a very large dielectric constant in PbTe of order 1000, we do not observe Coulomb blockade which typically obfuscates the orbital and spin spectra. We extract large and highly anisotropic effective Landé g-factors, in the range 20-44. The absence of Coulomb blockade allows direct readout, at zero source-drain bias, of spin-orbit hybridization energies of up to 600 \muμeV. These spin properties make PbTe nanowires, the recently synthesized members of group IV-VI materials family, attractive as a materials platform for quantum technology, such as spin and topological qubits.