Direct fluoride monitoring using a fluorogenic RNA-based biosensor.

Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

RNA quality score evaluation: A preliminary study of RNA integrity number (RIN) and RNA integrity and quality number (RNA IQ).

In the past several years, with the in-depth development of RNA-related research, exploring the application of transcriptome and corresponding RNA biomarkers has become one of the research hotspots in the field of forensic science. High-quality RNA is essential for successful downstream workflows, especially in the steps of screening biomarkers by microarray or RNA sequencing (RNA-seq). Thus, accurately evaluating the quality of RNA samples is a critical step in obtaining meaningful expression data. The RNA integrity number (RIN) generated from the Agilent Bioanalyzer system has been widely used for RNA quality control in the past two decades. Recently, Thermo Fisher Scientific launched a ratiometric fluorescence-based method to quickly check whether an RNA sample has degraded, and the results are presented as RNA integrity and quality number (RNA IQ). Both quality score systems determine RNA quality using a numerical system based on a scale of 1-10, with 1 denoting significantly degraded specimens and 10 representing high-quality, intact RNA samples. In this preliminary study, we evaluated the consistency, reproducibility and linearity of two quality scores in RNA quality determination by analyzing heat- and RNase- artificially degraded samples. Meanwhile, the expression levels of three microRNAs (hsa-let-7 g-5p, hsa-miR-93-5p and hsa-miR-191-5p) in intact and severely degraded RNA samples were estimated by TaqMan-qPCR and droplet digital PCR. Overall, both quality scores showed good repeatability and reproducibility in their respective tests. In the samples subjected to thermal degradation, RIN showed a trend corresponding to heating time, while RNA IQ value showed almost no change on the time gradient. However, in RNase A mediated degradation, RNA IQ value observed better linearity. Furthermore, the expression levels of three microRNAs in the severely degraded samples did not show significant changes compared to the intact RNA samples. RNA degradation is a very complex and highly variable process, which is difficult to comprehensively evaluate through any one index and cannot directly compare these two parameters. Nevertheless, combined with previous research results and the expression levels of three microRNAs in this study, analyzing RNA biomarkers with stable regions or small sizes in challenged samples may be a conservative and reliable approach.

Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer.

BACKGROUND: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance. MATERIALS AND METHODS: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations. RESULTS: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80). CONCLUSIONS: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer.

Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

Immunophenotypic and Gene Expression Analyses of the Inflammatory Microenvironment in High-Grade Oral Epithelial Dysplasia and Oral Lichen Planus.

BACKGROUND: Oral lichen planus (OLP) and oral epithelial dysplasia (OED) present diagnostic challenges due to clinical and histologic overlap. This study explores the immune microenvironment in OED, hypothesizing that immune signatures could aid in diagnostic differentiation and predict malignant transformation. METHODS: Tissue samples from OED and OLP cases were analyzed using immunofluorescence/immunohistochemistry (IF/IHC) for CD4, CD8, CD163/STAT1, and PD-1/PDL-1 expression. RNA-sequencing was performed on the samples, and data was subjected to CIBERSORTx analysis for immune cell composition. Gene Ontology analysis on the immune differentially expressed genes was also conducted. RESULTS: In OED, CD8 + T-cells infiltrated dysplastic epithelium, correlating with dysplasia severity. CD4 + lymphocytes increased in the basal layer. STAT1/CD163 + macrophages correlated with CD4 + intraepithelial distribution. PD-1/PDL-1 expression varied. IF/IHC analysis revealed differential immune cell composition between OED and OLP. RNA-sequencing identified upregulated genes associated with cytotoxic response and immunosurveillance in OED. Downregulated genes were linked to signaling, immune cell recruitment, and tumor suppression. CONCLUSIONS: The immune microenvironment distinguishes OED and OLP, suggesting diagnostic potential. Upregulated genes indicate cytotoxic immune response in OED. Downregulation of TRADD, CX3CL1, and ILI24 implies dysregulation in TNFR1 signaling, immune recruitment, and tumor suppression. This study contributes to the foundation for understanding immune interactions in OED and OLP, offering insights into future objective diagnostic avenues.

Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Mitochondria-Associated Transcriptome Profiling via Localizable Aggregation-Induced Emission Photosensitizers in Live Cells.

In recent decades, there has been increasing interest in studying mitochondria through transcriptomic research. Various exogenous fusion protein-based proximity labeling methods have been reported that focus on the site of one particular protein/peptide and might also influence the corresponding localization or interactome. To enable unbiased and high spatial-resolution profiling of mitochondria-associated transcriptomes in live cells, a flexible RNA proximity labeling approach was developed using aggregation-induced emission (AIE) type photosensitizers (PSs) that possess great mitochondria-targeting capabilities. Their accumulation in an enclosed mitochondrial environment tends to enhance the fluorescence emission and reactive oxygen species generation. By comparing the in vitro optical properties, photosensitization processes, as well as the in cellulo mitochondrial specificity and RNA labeling performance of four AIE PSs, high-throughput sequencing analysis was conducted using TFPy-mediated RNA proximity labeling in live HeLa cells. This approach successfully captured a comprehensive list of transcripts, including mitochondria-encoded RNAs, as well as some nuclear-derived RNAs located at the outer mitochondrial membrane and interacting organelles. This small molecule-based proximity labeling method bypasses complex genetic manipulation and transfection steps, making it readily applicable for diverse research purposes.

Self-Assembling Polypeptides in Complex Coacervation.

ConspectusIntracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular "condensates" playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation and dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA.Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations.Our laboratory has utilized complex coacervates assembled from oppositely charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules.Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such "guest" molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules, such as viral vaccines.Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates, while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning medicine to biocatalysis.

Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization.

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.

Influence of the isolation method on characteristics and functional activity of mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicle (EV) isolation methods are based on different physicochemical properties and may result in the purification of distinct EV populations. We compared two different isolation methods suitable for producing clinical-grade mesenchymal stromal cell-derived EVs (MSC-EVs)-ion exchange chromatography (IEX) and ultrafiltration (UF)-and evaluated their impact on the composition and functional properties of EVs. METHODS: EVs were purified from conditioned culture medium using an anion exchange resin (IEX) or Amicon filters with a 100-kDa cutoff (UF) (MilliporeSigma, Burlington, MA, USA). We assessed nanoparticle size and distribution by nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) and morphology by transmission electron microscopy. We also measured protein, lipid and total RNA concentration and immunophenotyped both EV populations by flow cytometry (MACSPlex assay; Miltenyi Biotec, Bergisch Gladbach, Germany). Moreover, immunomodulatory activity was tested using a standardized macrophage polarization assay and T-cell stimulation assay. Finally, proteomic analysis and cytokine quantification were carried out to better characterize both EV populations. RESULTS: We found by both TRPS and NTA that IEX and UF yielded a comparable amount of total particles with similar size and distribution. In addition, a similar quantity of lipids was obtained with the two procedures. However, IEX yielded 10-fold higher RNA quantity and a larger amount of proteins than UF. MSC-EVs isolated from IEX and UF were positive for the exosome markers CD9, CD63 and CD81 and showed a comparable surface marker expression pattern. Both populations demonstrated immunomodulatory activity in vitro, as they prevented acquisition of the M1 phenotype in lipopolysaccharide-stimulated macrophages and inhibited acquisition of the activation markers CD69 and CD25 on T cells, but the IEX-EVs exerted a significantly greater immunomodulatory effect on both macrophages and T cells compared with UF-EVs. Proteomic analysis and gene ontology enrichment analysis revealed no major differences between the preparations. Finally, cytokine quantification revealed that IEX-EVs were more enriched in some crucial anti-inflammatory and immunomodulatory cytokines (e.g., IL-2, IL-10, transforming growth factor beta and vascular endothelial growth factor) compared with UF-EVs. CONCLUSIONS: MSC-EVs isolated by IEX and UF displayed similar physicochemical, phenotypic and functional characteristics. In our conditions, both EV populations demonstrated important anti-inflammatory activity in macrophages and T cells. However, IEX-EVs were more potent than UF-EVs, which may indicate the superiority of this method for the production of clinical-grade EVs.

A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain.

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1-3. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets.

Numerous methodologies are used for blood RNA extraction, and large quantitative differences in recovered RNA content are reported. We evaluated three archived data sets to determine how extraction methodologies might influence mRNA and lncRNA sequencing results. The total quantity of RNA recovered /ml of blood affects RNA sequencing by impacting the recovery of weakly expressed mRNA, and lncRNA transcripts. Transcript expression (TPM counts) plotted in relation to transcript size (base pairs, bp) revealed a 30% loss of short to midsized transcripts in some data sets. Quantitative recovery of RNA is of considerable importance, and it should be viewed more judiciously. Transcripts common to the three data sets were subsequently normalized and transcript mean TPM counts and TPM count coefficient of variation (CV) were plotted in relation to increasing transcript size. Regression analysis of mean TPM counts versus transcript size revealed negative slopes in two of the three data sets suggesting a reduction of TPM transcript counts with increasing transcript size. In the third data set, the regression slope line of mRNA transcript TPM counts approximates zero and TPM counts increased in proportion to transcript size over a range of 200 to 30,000 bp. Similarly, transcript TPM count CV values also were uniformly distributed over the range of transcript sizes. In the other data sets, the regression CV slopes increased in relation to transcript size. The recovery of weakly expressed and /or short to midsized mRNA and lncRNA transcripts varies with different RNA extraction methodologies thereby altering the fundamental sequencing relationship between transcript size and TPM counts. Our analysis identifies differences in RNA sequencing results that are dependent upon the quantity of total RNA recovery from whole blood. We propose that incomplete RNA extraction directly impacts the recovery of mRNA and lncRNA transcripts from human blood and speculate these differences contribute to the "batch" effects commonly identified between sequencing results from different archived data sets.

Nascent ribosomal RNA act as surfactant that suppresses growth of fibrillar centers in nucleolus.

Liquid-liquid phase separation (LLPS) has been thought to be the biophysical principle governing the assembly of the multiphase structures of nucleoli, the site of ribosomal biogenesis. Condensates assembled through LLPS increase their sizes to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their sizes do not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the FC growth, we here construct a minimal theoretical model by taking into account nascent pre-rRNAs tethered to FC surfaces by RNA polymerase I. The prediction of this theory was supported by our experiments that quantitatively measure the dependence of the size of FCs on the transcription level. This work sheds light on the role of nascent RNAs in controlling the size of nuclear bodies.

The characterization of RNA-binding proteins and RNA metabolism-related proteins in fungal extracellular vesicles.

RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted to intracellular functions and can be found in extracellular vesicles (EVs) in different mammalian cells. EVs released by fungi contain a variety of proteins involved in RNA metabolism. These include RNA helicases, which play essential roles in RNA synthesis, folding, and degradation. Aminoacyl-tRNA synthetases, responsible for acetylating tRNA molecules, are also enriched in EVs, suggesting a possible link between these enzymes and tRNA fragments detected in EVs. Proteins with canonical RNA-binding domains interact with proteins and RNA, such as the RNA Recognition Motif (RRM), Zinc finger, and hnRNP K-homology (KH) domains. Polyadenylate-binding protein (PABP) plays a critical role in the regulation of gene expression by binding the poly(A) tail of messenger RNA (mRNA) and facilitating its translation, stability, and localization, making it a key factor in post-transcriptional control of gene expression. The presence of proteins related to the RNA life cycle in EVs from different fungal species suggests a conserved mechanism of EV cargo packing. Various models have been proposed for selecting RNA molecules for release into EVs. Still, the actual loading processes are unknown, and further molecular characterization of these proteins may provide insight into the mechanism of RNA sorting into EVs. This work reviews the current knowledge of RBPs and proteins related to RNA metabolism in EVs derived from distinct fungi species, and presents an analysis of proteomic datasets through GO term and orthology analysis, Our investigation identified orthologous proteins in fungal EVs on different fungal species.

Multitarget Stool RNA Test for Colorectal Cancer Screening.

Importance: Noninvasive tests for colorectal cancer screening must include sensitive detection of colorectal cancer and precancerous lesions. These tests must be validated for the intended-use population, which includes average-risk individuals 45 years or older. Objective: To evaluate the sensitivity and specificity of a noninvasive, multitarget stool RNA (mt-sRNA) test (ColoSense) test compared with results from a colonoscopy. Design, Setting, and Participants: This phase 3 clinical trial (CRC-PREVENT) was a blinded, prospective, cross-sectional study to support a premarket approval application for a class III medical device. A total of 8920 participants were identified online using social media platforms and enrolled from June 2021 to June 2022 using a decentralized nurse call center. All participants completed the mt-sRNA test, which incorporated a commercially available fecal immunochemical test (FIT), concentration of 8 RNA transcripts, and participant-reported smoking status. Stool samples were collected prior to participants completing a colonoscopy at their local endoscopy center. The mt-sRNA test results (positive or negative) were compared with index lesions observed on colonoscopy. Over the course of 12 months, individuals 45 years and older were enrolled in the clinical trial using the decentralized recruitment strategy. Participants were enrolled from 49 US states and obtained colonoscopies at more than 3800 different endoscopy centers. Main Outcomes and Measures: The primary outcomes included the sensitivity of the mt-sRNA test for detecting colorectal cancer and advanced adenomas and the specificity for no lesions on colonoscopy. Results: The mean (range) age of participants was 55 (45-90) years, with 4% self-identified as Asian, 11% as Black, and 7% as Hispanic. Of the 8920 eligible participants, 36 (0.40%) had colorectal cancer and 606 (6.8%) had advanced adenomas. The mt-sRNA test sensitivity for detecting colorectal cancer was 94%, sensitivity for detecting advanced adenomas was 46%, and specificity for no lesions on colonoscopy was 88%. The mt-sRNA test showed significant improvement in sensitivity for colorectal cancer (94% vs 78%; McNemar P = .01) and advanced adenomas (46% vs 29%; McNemar P < .001) compared with results of the FIT. Conclusions and Relevance: In individuals 45 years and older, the mt-sRNA test showed high sensitivity for colorectal neoplasia (colorectal cancer and advanced adenoma) with significant improvement in sensitivity relative to the FIT. Specificity for no lesions on colonoscopy was comparable to existing molecular diagnostic tests. Trial Registration: ClinicalTrials.gov Identifier: NCT04739722.

RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples.

Clinical tumor tissues that are preserved as formalin-fixed paraffin-embedded (FFPE) samples result in extensive cross-linking, fragmentation, and chemical modification of RNA, posing significant challenges for RNA-seq-based gene expression profiling. This study sought to define an optimal RNA-seq protocol for FFPE samples. We employed a common RNA extraction method and then compared RNA-seq library preparation protocols including RNAaccess, RiboZero and PolyA in terms of sequencing quality and concordance of gene expression using FFPE and case-matched fresh-frozen (FF) triple-negative breast cancer (TNBC) tissues. We found that RNAaccess, a method based on exome capture, produced the most concordant results. Applying RNAaccess to FFPE gastric cancer tissues, we established a minimum RNA DV200 requirement of 10% and a RNA input amount of 10ng that generated highly reproducible gene expression data. Lastly, we demonstrated that RNAaccess and NanoString platforms produced highly concordant expression profiles from FFPE samples for shared genes; however, RNA-seq may be preferred for clinical biomarker discovery work because of the broader coverage of the transcriptome. Taken together, these results support the selection of RNA-seq RNAaccess method for gene expression profiling of FFPE samples. The minimum requirements for RNA quality and input established here may allow for inclusion of clinical FFPE samples of sub-optimal quality in gene expression analyses and ultimately increasing the statistical power of such analyses.

Double blocking gap-filling-ligation coupled with cascade isothermal amplification for ultrasensitive quantification of N6-methyladenosine.

We developed a method for quantifying N6-methyladenosine at one-nucleotide resolution based on double blocking gap-filling-ligation and cascade isothermal amplification. This proposed method can detect as low as 1 fM target RNA, achieving selectivity up to approximately 100-fold between m6A and A, and has been successfully applied to the analysis of m6A at specific sites in cell samples.

A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes.

The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.