False Positive Signals Research Articles

Timing Matters: A Machine Learning Method for the Prioritization of Drug-Drug Interactions Through Signal Detection in the FDA Adverse Event Reporting System and Their Relationship with Time of Co-exposure.

Current drug-drug interaction (DDI) detection methods often miss the aspect of temporal plausibility, leading to false-positive disproportionality signals in spontaneous reporting system (SRS) databases. This study aims to develop a method for detecting and prioritizing temporally plausible disproportionality signals of DDIs in SRS databases by incorporating co-exposure time in disproportionality analysis. The method was tested in the Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS). The CRESCENDDI dataset of positive controls served as the primary source of true-positive DDIs. Disproportionality analysis was performed considering the time of co-exposure. Temporal plausibility was assessed using the flex point of cumulative reporting of disproportionality signals. Potential confounders were identified using a machine learning method (i.e. Lasso regression). Disproportionality analysis was conducted on 122 triplets with more than three cases, resulting in the prioritization of 61 disproportionality signals (50.0%) involving 13 adverse events, with 61.5% of these included in the European Medicine Agency's (EMA's) Important Medical Event (IME) list. A total of 27 signals (44.3%) had at least ten cases reporting the triplet of interest, and most of them (n = 19; 70.4%) were temporally plausible. The retrieved confounders were mainly other concomitant drugs. Our method was able to prioritize disproportionality signals with temporal plausibility. This finding suggests a potential for our method in pinpointing signals that are more likely to be furtherly validated.

Pt–S bond stabilized DNAzyme nanosensor with thiol-resistance enabling high-fidelity biosensing

Gold nanoparticles (Au NPs) have been widely utilized in developing DNAzyme-functionalized nanosensors, most of which were engineered by attaching the thiolated DNAzymes to Au NPs via Au–S bonding. However, the Au NP-DNAzyme nanosensors always suffer from signal distortion when applied in complex environment with abundant thiols, which poses challenge for practical applications. Here, we focus on addressing the root cause of the issue and propose to decorate the Au NPs with a thin layer of platinum, thus facilitating the conjugation of DNAzymes through Pt–S bonding, a thiol-resistant cross-linking. The Pt–S bond stabilized DNAzyme nanosensor effectively minimized false positive signals when detecting l-histidine in infant formulas, as compared to the Au–S stabilized counterpart. This innovative strategy holds promise for high-fidelity biosensing, improving the practical applicability of Au NP-based DNAzyme nanosensor.

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/μL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

Strain-specific and sensitive monitoring of the biocontrol agent Trichoderma asperellum T34 in growing medium via real-time PCR

ABSTRACT Trichoderma asperellum Samuels, Lieckf. & Nirenberg is one of several Trichoderma species comprising effective biocontrol strains. One of these, Trichoderma asperellum T34, shows activity against soilborne pathogens such as Rhizoctonia, Fusarium, and Pythium. The activity of biocontrol strains such as T34 depends on their establishment in the rhizosphere. Monitoring of the population of T34 and other strains in the rhizosphere and the growing medium is therefore important for evaluating their potential in different crops and different growing systems. Until now, strain-specific monitoring has been very difficult as the main quantification methods, i.e. traditional dilution plating or standard molecular detection methods, are specific only at genus or species level, respectively. Genotyping-by-sequencing was used to identify genomic loci specific to T34. These were used to develop strain-specific Taqman real-time (qPCR) assays. After testing for specificity and sensitivity, two assays were combined in duplex format. The duplicate assay provided an internal control to avoid a false positive signal in case one of the target loci would be present in an as yet unidentified non-target organism. An optional enrichment step in semi-selective medium was included for increased sensitivity in cases of small T34 population size, theoretically allowing detection down to 0.1 spore equivalent per mL growing medium. The techniques were applied to 200 mL samples of growing medium analyzed 0, 19, and 112 days after spiking with different numbers and combinations of spores from target and non-target Trichoderma isolates. This technique shows potential for developing other strain-specific detection assays using an affordable genotyping tool.

False Positive Signals in the Detection of Explosives by Ion Mobility Spectrometry: Organic Acids

False Positive Signals in the Detection of Explosives by Ion Mobility Spectrometry: Organic Acids

Reliable Identification of Binary Supermassive Black Holes from Rubin Observatory Time-domain Monitoring

Periodic signatures in time-domain observations of quasars have been used to search for binary supermassive black holes (SMBHs). These searches, across existing time-domain surveys, have produced several hundred candidates. The general stochastic variability of quasars, however, can masquerade as a false-positive periodic signal, especially when monitoring cadence and duration are limited. In this work, we predict the detectability of binary SMBHs in the upcoming Rubin Observatory Legacy Survey of Space and Time (LSST). We apply computationally inexpensive sinusoidal curve fits to millions of simulated LSST Deep Drilling Field light curves of both single, isolated quasars and binary quasars. The period and phase of simulated binary signals can generally be disentangled from quasar variability. Binary amplitude is overestimated and poorly recovered for two-thirds of potential binaries due to quasar accretion variability. Quasars with strong intrinsic variability can obscure a binary signal too much for recovery. We also find that the most luminous quasars mimic current binary candidate light curves and their properties: The false-positive rates are 60% for these quasars. The reliable recovery of binary period and phase for a wide range of input binary LSST light curves is promising for multi-messenger characterization of binary SMBHs. However, pure electromagnetic detections of binaries using photometric periodicity with amplitude greater than 0.1 mag will result in samples that are overwhelmed by false positives. This paper represents an important and computationally inexpensive way forward for understanding the true and false-positive rates for binary candidates identified by Rubin.

Abstract 1035: Identification of fibroblast growth factor receptors (FGFRs) alterations (alts) at DNA and RNA-level by one-step next-generation sequencing (NGS) panel

Abstract Background: FGFR inhibitors are currently in clinical development or approved for urothelial cancer and cholangiocarcinoma with FGFR fusion (FUS) or/and mutation (MUT). AmoyDx FGFR 1-4 NGS Panel (FGFR-P) was developed for FGFR alts detection based on both DNA and RNA. The goal of this study is to validate and assess the performance of FGFR-P in pan-cancer FGFR alts detection by comparing with an AmoyDx comprehensive genomic profiling test (CGP) and a health authority approved DNA-based NGS panel (DNA-P). Methods: Both FGFR-P and CGP are DNA (MUT) and RNA (FUS) based for detecting FGFR alts with optimized bioinformatics pipeline to eliminate baseline noise caused by deamination events, which is commonly found in aged FFPE samples. Cell lines, cell line/patient derived xenografts (CDXs/PDXs) and 397 samples of 26 cancer types (90 samples >10 years) were used for validation of FGFR-P. Total 382 and 50 pan-cancer samples were respectively tested to compare FGFR-P with CGP and DNA-P. Results: To validate accuracy, 36 FFPE samples from cell lines and CDXs/PDXs with known FGFR status (25 FUSs, 5 MUTs, 6 wild-types) were tested by FGFR-P with concordance rate 100%. In clinical sample testing, FGFR-P also showed high agreement with CGP (99.5%) and DNA-P (98.0%). Conflicting result confirmed by FISH revealed 1 FUS missed by DNA-P but detected by FGFR-P, which demonstrated advantage of FGFR alts detection by DNA+RNA. In addition, an optimized bioinformatics pipeline by mimic samples with deamination events effectively filtered out C:G>T:A false positive signals. FGFR-P achieved high success rate in testing pan-tumor samples (89.9%) including samples >10 years (75.6%). Conclusions: FGFR-P provides a novel opportunity to identify FGFR alt pan-cancer patients using a robust DNA+RNA NGS platform, which shows high success rate even in aged samples and will be a potent tool for sensitive and reliable detection of FGFR alts for clinical diagnostics. CGP FGFR-P MUT Positive Negative Total Positive 29 0 29 Negative 1 194 195 Total 30 194 224 FUS Positive Negative Total Positive 20 1 21 Negative 0 137 137 Total 20 138 158 The overall percent agreement 99.5% Citation Format: Min Qing, Xuejun Chen, Wenqing Su, Xiaofang Zhuo, Ting Shen, Chengjuan Xiong, Xuesong Lyu, Renee Tate, Qibiao Wu, Longen Zhou, Shibu Thomas, Wangwang Ning, Jianqing Wang, Huihui Yan, Zhiqiang Yin, Zhan Huang, Yaxin Xue, Changbin Zhu. Identification of fibroblast growth factor receptors (FGFRs) alterations (alts) at DNA and RNA-level by one-step next-generation sequencing (NGS) panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1035.

Abstract 2323: Deciphering RNA degradation: Insights from a comparative analysis of paired fresh frozen/FFPE total RNA-seq

Abstract Background: Fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples are primary resources for archival tissues in cancer studies. Despite the advantages of easy storage and cost-effectiveness, FFPE samples have the disadvantage of inevitable chemical-induced RNA degradation. Although, in mRNA-seq data, the 3' bias due to the characteristics of the mRNA-seq platform allows the measure of RNA degradation levels, for total RNA-seq and FFPE samples, there is still no clear measure for RNA degradation. Methods: Our study, utilizing The Cancer Genome Atlas (TCGA) pilot data comprised of 26 paired samples of fresh frozen mRNA-seq [FFM], fresh frozen total RNA-seq [FFT], and FFPE total RNA-seq [PET], investigated RNA degradation patterns in FFPE samples. Upon investigating the read coverage of transcripts in FFPE data, we observed increased noise parameters in selected samples compared to others, suggesting a method for assessment of RNA degradation in FFPE. To measure these noise patterns, we developed a method called windowCV (wCV) utilizing coefficient of variance (CV) along the transcript length. Also, based on reports that membrane-surrounded RNA such as lncRNA and mitochondrial RNA (mtRNA) might be less affected by formalin fixation, we developed the simple measure dividing expression value of other proteins with certain lncRNA or mtRNA inferring the degree of RNA degradation. Results: We first compared the shape of transcript read coverage from 3 different RNA-seq data and observed 3’bias pattern in FFM which was not observed in FFT and PET. For FFT and PET, we applied wCV and executed hierarchical clustering. We observed that a subset of samples and genes were clustered by high wCV value suggesting notable RNA degradation. To support the hypothesis of RNA degradation, we utilized Trimmed Mean of M values (TMM) expression ratio of paired FFT and PET to determine which samples and genes were associated with underestimation of expression in PET. Interestingly, we observed that PET/FFT TMM ratio showed negative correlation with wCV. Additionally, when we investigated hierarchical clustering result of PET/FFT TMM ratio, we found that lncRNA and mtRNA were less affected by FFPE. We then calculated expression ratio between membrane-surrounded RNA and a set of protein coding genes largely affected by FFPE. As a result, we found that this ratio positively correlated with paired PET/FFT TMM ratio. We conclude that this simple measurement can provide information of FFPE induced underestimated expression with limited transcriptome data as well as RNA degradation. Conclusion: Our method can provide gene-level CV while avoiding false positive variance signal originating from transcript model and simple way to detect the degree of RNA degradation across FFPE samples. Citation Format: Wonyoung Choi, Miyeon Yeon, Hyo Young Choi, David Neil Hayes. Deciphering RNA degradation: Insights from a comparative analysis of paired fresh frozen/FFPE total RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2323.

Abstract 724: MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling

Abstract The development of novel antibody drugs is a lengthy journey. Many factors can lead to failures during clinical development. According to reports, off-target binding of antibodies could also be a significant cause. It has been stated that about 25% of preclinical candidates might have off-target issues. With the emergence of new modalities like CD3 bispecific antibodies, ADCs, and CAR-T therapies, early-stage off-target screening of antibodies has become even more crucial. Although techniques such as immunohistochemistry, ELISA, and flow cytometry (FACS) have been used for off-target testing of antibodies, these methods still lack sensitivity and specificity, and their throughput is limited. To address the need for antibody target deconvolution and receptor identification, we developed a novel membrane protein screening array-AB5000 (MPSA-AB5000). MPSA-AB5000 is a high-throughput cell-based platform for identifying the targets of antibodies and other ligands that bind to membrane proteins. MPSA-AB5000 is an excellent solution for antibody specificity screening, which enables early off-target screening and can de-risk the antibody drug development program. The MPSA-AB5000 makes use of the full coverage of unique human membrane proteins, including receptors, transporters, enzymes, miscellaneous, and others. Each membrane protein is expressed in live cells to ensure native conformation. In addition, the same membrane protein clones with a C-terminal epitope tag were constructed, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, MPSA-AB5000 uses luciferase reporter cell line detection. The wash-free strategy reduces false negatives, and signal amplification makes remarkable discrimination. Compared to similar technologies, we possess significant advantages in the following aspects: a. High sensitivity and sigh throughput: our technology platform offers not only high sensitivity, capable of detecting subtle interactions but also supports high-throughput screening, allowing for the evaluation of a large number of samples in a short timeframe. b. Avoidance of false negative signals: by eliminating washing steps, our method reduces the risk of false negative signals, ensuring reliable detection of non-specific binding. c. Comprehensive validation approaches: We employ various validation methods such as FACS and ELISA to eliminate false positive signals and enhance the accuracy and credibility of our results. All the advantages ensure that MPSA-AB5000 is more rapid, simple, and highly sensitive than traditional assays. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 724.

Pt-Decorated Gold Nanoflares for High-Fidelity Phototheranostics: Reducing Side-Effects and Enhancing Cytotoxicity toward Target Cells.

Functionalized with the Au-S bond, gold nanoflares have emerged as promising candidates for theranostics. However, the presence of intracellular abundantly biothiols compromises the conventional Au-S bond, leading to the unintended release of cargoes and associated side-effects on non-target cells. Additionally, the hypoxic microenvironment in diseased regions limits treatment efficacy, especially in photodynamic therapy. To address these challenges, high-fidelity photodynamic nanoflares constructed on Pt-coated gold nanoparticles (Au@Pt PDNF) were communicated to avoid false-positive therapeutic signals and side-effects caused by biothiol perturbation. Compared with conventional photodynamic gold nanoflares (AuNP PDNF), the Au@Pt PDNF were selectively activated by cancer biomarkers and exhibited high-fidelity phototheranostics while reducing side-effects. Furthermore, the ultrathin Pt-shell catalysis was confirmed to generate oxygen which alleviated hypoxia-related photodynamic resistance and enhanced the antitumor effect. This design might open a new venue to advance theranostics performance and is adaptable to other theranostic nanomaterials by simply adding a Pt shell.

Monitoring the Intracellular Fate of Molecular Beacons: The Challenge of False Positive Signals

Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time‐stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta‐cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant.

Unique fluorescent probe for the recognition of late apoptosis via translocation from plasma membrane to nucleus

Cell apoptosis is a crucial physiological process playing central roles in key biological and pathological activities. However, the current fluorescent probes for the detection of late apoptosis were “off–on” probes, which were facilely interfered by false positive signals caused by inhomogeneous staining and other factors. Herein, a unique fluorescent probe (NPn) discriminating late apoptosis from early apoptosis and heathy status with two different sets of fluorescent signals have been prepared, to overcome the possible false positive signals. NPn was designed impermeable to biomembranes and simultaneously with high affinity to DNA/RNA, which localized on the plasma membranes of living and early apoptotic cells, while relocated to the nucleus in late apoptotic cells. The hydrophilic amine unit and small ion radius were responsive for its membrane impermeability, which was confirmed with two control molecules without amine group. Using the probe, we have successfully evaluated the cell apoptosis induced by ultraviolet irradiation, rotenone, colchicine, and paclitaxel, demonstrating its potential application in biological researches.

CRISPR-Cas12a based target recognition initiated duplex-specific nuclease enhanced fluorescence and colorimetric analysis of cell-free DNA (cfDNA)

The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers.

Self-evolving persistent luminescence nanoprobes for autofluorescence-free ratiometric imaging and on-demand enhanced chemodynamic therapy of pulmonary metastatic tumors.

Precise imaging-guided therapy of a pulmonary metastasis tumor is of great significance for tumor management and prognosis. Persistent luminescence nanoparticles (PLNPs) are promising probes due to their in situ excitation-free and low-background imaging characteristics. However, most of the PLNP-based probes cannot intelligently distinguish between normal and tumor tissues or balance the needs of targeted accumulation and rapid metabolism, resulting in false positive signals and potential side effects. Besides, the luminescence intensity of single-emissive PLNPs is affected by external factors. Herein, we report a self-evolving double-emissive PLNP-based nanoprobe ZGMC@ZGC-TAT for pulmonary metastatic tumor imaging and therapy. Acid-degradable green-emitting PLNPs (ZGMC) with good afterglow performance and therapeutic potential are synthesized by systematic optimization of dopants. Ultra-small red-emitting PLNPs (ZGC) are then prepared as imaging and reference probes. The two PLNPs are finally covalently coupled and further modified with a cell-penetrating peptide (TAT) to obtain ZGMC@ZGC-TAT. Dual emission ensures a stable luminescence ratio (I700/I537) independent of probe concentration, test voltage and time gate. ZGMC degrades and phosphorescence disappears in a tumor microenvironment (TME), resulting in an increase in I700/I537, thus enabling tumor-specific ratiometric imaging. Cu2+ and Mn2+ released by ZGMC degradation achieve GSH depletion and enhance CDT, effectively inhibiting tumor cell proliferation. Meanwhile, the size of ZGMC@ZGC-TAT decreases sharply, and the resulting ZGC-TAT further causes nuclear pyknosis and quickly clear metabolism. The developed ZGMC@ZGC-TAT turns non-targeted lung aggregation of nanomaterials into a unique advantage, and integrates TME-triggered phosphorescence and size self-evolution, and on-demand therapeutic functions, showing outstanding prospects in precise imaging and efficient treatment of pulmonary metastatic tumors.

Rapid detection of HPV16 utilizing recombinase polymerase amplification with the employment of an extremely low concentration of the probe.

Human papillomavirus 16 (HPV16) infection is the leading cause of cervical cancer. The current mainstream method for detecting HPV16 is quantitative real-time PCR (qPCR). However, due to its time-consuming nature and reliance on expensive qPCR instruments, there is growing interest in more convenient, rapid and private approaches such as recombinase polymerase amplification (RPA). In this study, we innovated an effective method for HPV16 detection by integrating a RPA system with lateral flow strip (LFS) detection. Primers with optimal efficiency and specificity were designed, and false positive signals caused by dimers were eliminated by reducing the probe concentration. The RPA-LFS method demonstrates high sensitivity and specificity, capable of detecting 230 copies per reaction of HPV16 within 25 min without cross-reactivity to other subtypes. It exhibits good tolerance, remaining unaffected by 1.0% miconazole, 0.5% tioconazole and 1.0% hemoglobin. The results of clinical samples detected by this method were consistent with those of qPCR. The method provides a practical reference for HPV16 diagnosis and can be valuable in home and resource-limited settings, contributing to the reduction of cervical cancer incidence.

Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation.

Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis. Herein, we report a design strategy for probes that suppress undesired organelle accumulation using a bioisostere for intracellular protein imaging in living cells. Our design allows the protein labeling probes to possess both membrane permeability and suppress non-specific accumulation and has been shown to use several protein labeling systems, such as PYP-tag and Halo tag systems. We further developed a fluorogenic PYP-tag labeling probe for intracellular proteins and used it to visualize multiple localizations of target proteins in the intracellular system. Our strategy offers a versatile design for undesired accumulation-suppressed probes with cationic dye scaffolds and provides a valuable tool for intracellular protein imaging.

Biotin-functionalized nanoparticles: an overview of recent trends in cancer detection.

Electrochemical bio-sensing is a potent and efficient method for converting various biological recognition events into voltage, current, and impedance electrical signals. Biochemical sensors are now a common part of medical applications, such as detecting blood glucose levels, detecting food pathogens, and detecting specific cancers. As an exciting feature, bio-affinity couples, such as proteins with aptamers, ligands, paired nucleotides, and antibodies with antigens, are commonly used as bio-sensitive elements in electrochemical biosensors. Biotin-avidin interactions have been utilized for various purposes in recent years, such as targeting drugs, diagnosing clinically, labeling immunologically, biotechnology, biomedical engineering, and separating or purifying biomolecular compounds. The interaction between biotin and avidin is widely regarded as one of the most robust and reliable noncovalent interactions due to its high bi-affinity and ability to remain selective and accurate under various reaction conditions and bio-molecular attachments. More recently, there have been numerous attempts to develop electrochemical sensors to sense circulating cancer cells and the measurement of intracellular levels of protein thiols, formaldehyde, vitamin-targeted polymers, huwentoxin-I, anti-human antibodies, and a variety of tumor markers (including alpha-fetoprotein, epidermal growth factor receptor, prostate-specific Ag, carcinoembryonic Ag, cancer antigen 125, cancer antigen 15-3, etc.). Still, the non-specific binding of biotin to endogenous biotin-binding proteins present in biological samples can result in false-positive signals and hinder the accurate detection of cancer biomarkers. This review summarizes various categories of biotin-functional nanoparticles designed to detect such biomarkers and highlights some challenges in using them as diagnostic tools.

Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Comparative analysis of micronucleus induction and DNA damage biomarkers in TK6 and A375 cells using flow cytometry.

Previously, we introduced an alternative adherent A375 cell line for clastogenicity and aneugenicity testing using a high content imaging platform. To further characterize the performance of A375 cells, we investigated the sensitivity and specificity of A375 and TK6 cells by directly comparing micronucleus (MN) induction, cytotoxicity (relative cell counts, viability, and apoptosis), clastogenicity (γH2AX), and aneuploidy markers (pH 3, MPM-2, and polyploidy) using flow cytometric methods. We evaluated 14 compounds across different mechanisms (non-genotoxic apoptosis inducers, clastogens, and aneugens with either tubulin binding or aurora kinase inhibiting phenotypes) at 4-h and 24-h post treatment. Both aneugens and clastogens tested positive for micronucleus induction in both cell lines. Apoptosis continued to be a confounding factor for flow cytometry-based micronuclei assessment in TK6 cells as evidenced by positive responses by the three cytotoxicants. Conversely, A375 cells were not affected by apoptosis-related false positive signals and did not produce a positive response in the in vitro micronucleus assay. Benchmark dose response (BMD) analysis showed that the induction of micronuclei and biomarkers occurred at similar concentrations in both cell lines for clastogens and aneugens. By showing that A375 cells have similar sensitivity to TK6 cells but a greater specificity, these results provide additional support for A375 cells to be used as an alternative adherent cell line for in vitro genetic toxicology assessment.