miRNA Concentrations Research Articles

Early Diagnosis of Triple-Negative Breast Cancer Based on Dual microRNA Detection Using a Well-Defined DNA Crown-Carbon Dots Structure as an Electrochemiluminescence Sensing Platform.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Thus, early detection and accurate diagnosis of this cancer are crucial for improving the survival rate of patients. Specific microRNAs (miRNAs) have been implicated in the occurrence, proliferation, and metastasis of TNBC. Addressing this need, our study developed a biosensor platform for early and accurate TNBC diagnosis by integrating electrochemiluminescence (ECL) technology with a DNA sensing strategy. Specifically, synthesized positively charged carbon dots (CDs) were used to neutralize the electrostatic repulsion between DNA strands and facilitate the assembly of DNA triangular prisms (DNA TP-CDs). Hairpins were then incorporated into the DNA TP-CDs to form the final DNA crown structure. The early TNBC biomarker, microRNA-93-3p (miR-93-3p), allowed for the binding between the DNA Crown and the DNA track on the electrode and initiated the ECL signal. Subsequently, microRNA-210 (miR-210) unlocked the DNA tripedal walker, and its movement on the DNA Crown eventually quenched the ECL signal, enabling accurate TNBC diagnosis and tumor stage assessment. Our proposed biosensor had satisfactory sensing efficiency due to the ordered DNA track and rapid-moving DNA walker. The data revealed a good linear relationship between the ECL signals and the logarithm of miRNA concentrations, with miR-93-3p having a detection limit of 31.04 aM and miR-210 having a detection limit of 7.69 aM. The biosensor also showed satisfactory performance in serum samples and cells. Taken together, this study hopes to provide ideas and applications for clinical diagnosis as well as the personalized treatment of TNBC.

Simultaneous Multi-miRNA Detection in Urinary Small Extracellular Vesicles Using Target-Triggered Locked Hairpin DNA-Functionalized Au Nanoprobes for Systemic Lupus Erythematosus Diagnosis.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by multiorgan involvement and complex clinical manifestations, leading to cumbersome diagnostic processes. MicroRNAs (miRNAs) in small extracellular vesicles (sEVs) have emerged as promising biomarkers for liquid biopsy. Herein, we constructed a simple multi-miRNA detection platform based on target-triggered locked hairpin DNA-functionalized Au nanoprobes (AuNP@LH) as a simple and noninvasive tool for the diagnosis and classification of SLE. The nanoprobes were prepared by modifying locked hairpin DNA designed for target miRNAs on gold nanoparticles. In the presence of target miRNAs, target-triggered hairpin assembly amplification was induced, and then fluorophore-labeled bolt DNA was released, resulting in a fluorescence signal responsive to miRNA concentration. Benefiting from the enzyme-free amplification strategy, the limits of detection (LOD) of three miRNA biomarkers for SLE were 19 pM for microRNA-146a, 66 pM for microRNA-29c, and 19 pM for microRNA-150. The proposed probes have been successfully applied to simultaneously detect multiple miRNAs in urinary sEVs from patients diagnosed with SLE and healthy controls, which exhibited good practicability in SLE diagnosis with the area under curve (AUC) of the receiver characteristic curve reaching 1.00. Furthermore, SLE patients with different disease severity can be differentiated with 81.2% accuracy. Predictably, with the advantages of low cost, rapidity, high sensitivity, and noninvasiveness, our multi-miRNA detection platform is a potential tool for multiple miRNA analysis and related clinical applications.

Development of a Modular miRNA-Responsive Biosensor for Organ-Specific Evaluation of Liver Injury.

MicroRNAs (miRNAs) are increasingly being considered essential diagnostic biomarkers and therapeutic targets for multiple diseases. In recent years, researchers have emphasized the need to develop probes that can harness extracellular miRNAs as input signals for disease diagnostics. In this study, we introduce a novel miRNA-responsive biosensor (miR-RBS) designed to achieve highly sensitive and specific detection of miRNAs, with a particular focus on targeted organ-specific visualization. The miR-RBS employs a Y-structured triple-stranded DNA probe (Y-TSDP) that exhibits a fluorescence-quenched state under normal physiological conditions. The probe switches to an activated state with fluorescence signals in the presence of high miRNA concentrations, enabling rapid and accurate disease reporting. Moreover, the miR-RBS probe had a modular design, with a fluorescence-labeled strand equipped with a functional module that facilitates specific binding to organs that express high levels of the target receptors. This allowed the customization of miRNA detection and cell targeting using aptameric anchors. In a drug-induced liver injury model, the results demonstrate that the miR-RBS probe effectively visualized miR-122 levels, suggesting it has good potential for disease diagnosis and organ-specific imaging. Together, this innovative biosensor provides a versatile tool for the early detection and monitoring of diseases through miRNA-based biomarkers.

Cyclic Enzymatic Signal Amplification-Driven DNA Logic Nanodevices on Framework Nucleic Acid for Highly Sensitive Electrochemiluminescence Detection of Dual Myocardial miRNAs.

MicroRNAs (miRNAs) have emerged as promising biomarkers for acute myocardial infarction (AMI). There is an urgent imperative to develop analytical methodologies capable of intelligently discerning multiple circulating miRNAs. Here, we present a dual miRNA detection platform for AMI using DNA logic gates coupled with an electrochemiluminescence (ECL) response. The platform integrates DNA truncated square pyramids as capture probes on gold-deposited electrodes, enabling precise quantification of miRNA associated with AMI. The cyclic enzymatic signal amplification principle of strand displacement amplification enhances the miRNA detection sensitivity. AND and OR logic gates have been successfully constructed, enabling intelligent identification of miRNAs in AMI. Calibration curves show strong linear correlations between ECL intensity and target miRNA concentration (10 fM to 10 nM), with excellent stability in consecutive measurements. When applied to clinical serum samples, the biosensor exhibits consistent performance, underscoring its reliability for clinical diagnostics. This innovative approach not only demonstrates DNA nanotechnology's potential in biosensing but also offers a promising solution for improving AMI diagnosis and prognosis through precise miRNA biomarker detection.

Potential diagnostic and prognostic biomarkers of pediatric Burkitt lymphoma identified through miRNA expression profiling.

Pediatric Burkitt lymphoma (pBL) is the most common non-Hodgkin lymphoma in children. These patients require prompt diagnosis and initiation of therapy due to rapid tumor growth. The roles of tumor tissue and circulating microRNAs (miRNAs) in the diagnosis or prognostication have not been fully elucidated in pBLs. Differentially expressed (DE) miRNAs were identified with microRNA sequencing (miRNA-Seq) in tumor tissues and plasma of diagnostic pBLs. The diagnostic potential of total miRNA concentrations and overexpressed miRNAs were evaluated through receiver operating characteristic (ROC) analyses. Log-rank test was employed to evaluate survival differences associated with DE miRNAs. Selected miRNA expressions were cross-validated with quantitative reverse transcription PCR (qRT-PCR). Total circulating cell-free miRNAs were higher in pBL cases compared to controls. Cancer-associated pathways were enriched among miRNAs differentially expressed in pBL tumor tissues. Several upregulated miRNAs in pBL tumors demonstrated high diagnostic potential. Similarly, ROC analysis of overexpressed plasma miRNAs revealed circulating cell-free or exosomal miRNAs that can distinguish pBLs from control cases. Indeed, integrative analysis of overexpressed circulating exosomal miRNAs showed an enhanced diagnostic potential for certain triple combinations. Kaplan-Meier analyses of DE miRNAs in tumor tissues identified miRNAs predicting overall survival. Differentially expressed miRNAs in tumor tissue and plasma of pBL have the potential to improve diagnosis and prognosis. Differentially expressed miRNAs in treatment-naive pediatric Burkitt lymphoma cases have diagnostic or prognostic biomarker potential. This is the first study that applied miRNA-Seq on treatment-naive pediatric Burkitt lymphoma cases for identification of differentially expressed miRNAs both in tumor tissue and plasma samples with diagnostic potential. Through systematic analysis of differentially expressed miRNAs, tumor tissue miRNAs associated with the overall survival of pBLs have been discovered. The clinically significant, differentially expressed miRNAs identified in pediatric Burkitt lymphoma cases can potentially improve the current tissue-based or non-invasive clinical practice in terms of diagnosis or prognostication.

Circulating miRNAs in the Plasma of Post-COVID-19 Patients with Typical Recovery and Those with Long-COVID Symptoms: Regulation of Immune Response-Associated Pathways.

Following the acute phase of SARS-CoV-2 infection, certain individuals experience persistent symptoms referred to as long COVID. This study analyzed the patients categorized into three distinct groups: (1) individuals presenting rheumatological symptoms associated with long COVID, (2) patients who have successfully recovered from COVID-19, and (3) donors who have never contracted COVID-19. A notable decline in the expression of miR-200c-3p, miR-766-3p, and miR-142-3p was identified among patients exhibiting rheumatological symptoms of long COVID. The highest concentration of miR-142-3p was found in healthy donors. One potential way to reduce miRNA concentrations is through antibody-mediated hydrolysis. Not only can antibodies possessing RNA-hydrolyzing activity recognize the miRNA substrate specifically, but they also catalyze its hydrolysis. The analysis of the catalytic activity of plasma antibodies revealed that antibodies from patients with long COVID demonstrated lower hydrolysis activity against five fluorescently labeled oligonucleotide sequences corresponding to the Flu-miR-146b-5p, Flu-miR-766-3p, Flu-miR-4742-3p, and Flu-miR-142-3p miRNAs and increased activity against the Flu-miR-378a-3p miRNA compared to other patient groups. The changes in miRNA concentrations and antibody-mediated hydrolysis of miRNAs are assumed to have a complex regulatory mechanism that is linked to gene pathways associated with the immune system. We demonstrate that all six miRNAs under analysis are associated with a large number of signaling pathways associated with immune response-associated pathways.

Serum miR-34a-5p, miR-103a-3p, and miR-376a-3p as possible biomarkers of conversion from relapsing-remitting to secondary progressive multiple sclerosis

Relapsing-remitting (RR) Multiple Sclerosis (MS) is the most common form of the disease; RRMS patients can maintain their clinical phenotype throughout life or can develop a secondary progressive (SP) course over time. We investigated whether circulating miRNAs can predict RR-to-SPMS conversion. A serum miRNAs profile was initially analyzed in a cross-sectional study by qPCR in 16 patients (8 RRMS and 8 SPMS) (Discovery cohort). Three miRNAs, i.e. miR-34a-5p, miR-103a-3p and miR-376a-3p, were significantly up-regulated in SPMS compared to RRMS patients (p < 0.0 5). Serum concentration of the same miRNAs was subsequently analyzed in a retrospective study by ddPCR at baseline in 69 RRMS patients who did (N = 36 cSPMS) or did not (N = 33) convert into SPMS over a 10-year observation period (Study cohort). The results showed that these miRNAs were significantly increased at baseline only in those RRMS patients who converted to SPMS over time. miR-34a-5p and miR-376a-3p alone were significantly increased in cSPMS sera at the end of the 10-years period too. Serum concentration of miR-34a-5p, miR-103a-3p and miR-376a-3p is increased in RRMS patients several years before their conversion to SPMS. These miRNAs might be useful biomarkers to predict the conversion from RRMS to SPMS.

Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.

Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.

Reduced ovarian cholesterol and steroid biosynthesis along with increased inflammation are associated with high DEHP metabolite levels in human ovarian follicular fluids

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to have endocrine-disrupting properties mediated by its many metabolites that form upon exposure in biological systems. In a previous study, we reported an inverse association between DEHP metabolites in the human ovarian follicular fluid (FF) and the responsiveness of the follicles to controlled ovarian stimulation during in vitro fertilization (IVF) treatments. Here, we explored this association further through molecular analysis of the ovarian FF samples.Ninety-six IVF patients from Swedish (N = 48) and Estonian (N = 48) infertility clinics were selected from the previous cohort (N = 333) based on the molar sum of DEHP metabolites in their FF samples to arrive at “high” (mean 7.7 ± SD 2.3 nM, N = 48) and “low” (0.8 ± 0.4 nM, N = 48) exposure groups. Extracellular miRNA levels and concentrations of 15 steroid hormones were measured across FF samples. In addition, FF somatic cells, available for the Estonian patients, were used for RNA sequencing.Differential expression (DE) and interactions between miRNA and mRNA networks revealed that the expression levels of genes in the cholesterol biosynthesis and steroidogenesis pathways were significantly decreased in the high compared to the low DEHP group. In addition, the DE miRNAs were predicted to target key enzymes within these pathways (FDR < 0.05). A decreased 17-OH-progesterone to progesterone ratio was observed in the FF of the high DEHP group (p < 0.05). Additionally, the expression levels of genes associated with inflammatory processes were elevated in the FF somatic cells, and a computational cell-type deconvolution analysis suggested an increased immune cell infiltration into the high DEHP follicles (p < 0.05).In conclusion, elevated DEHP levels in FF were associated with a significantly altered follicular milieu within human ovaries, involving a pro-inflammatory environment and reduced cholesterol metabolism, including steroid synthesis. These results contribute to our understanding of the molecular mechanisms of female reprotoxic effects of DEHP.

MiRNA-mediated insect-resistant transgenic rice poses no risk to a non-target parasitoid, Cotesia chilonis, via direct feeding or through its target host.

MicroRNAs (miRNAs) have started to play an important role in pest control, and novel miRNA-based transgenic insect-resistant plants are now emerging. However, an environmental risk assessment of these novel transgenic plants expressing insect miRNAs must be undertaken before promoting their application. Here, transgenic miR-14 rice, which has high resistance to the rice stem borer Chilo suppressalis, was used as an example for evaluation in this study. Taking the tier1 risk assessment method in Bacillus thuringiensis (Bt) crops as a reference, the effects of the direct exposure of a non-target parasitoid, Cotesia chilonis, to a high concentration of miRNA were evaluated. The results showed that direct feeding with miR-14 at high concentration had no significant effects on the biological parameters of Co.chilonis, whereas when miR-14 was injected into Ch.suppressalis-parasitized larvae, the development duration of Co.chilonis was significantly affected. In combination with the real conditions of the rice paddy field, it could be inferred that transgenic miR-14 rice has no significant negative effects on the important non-target parasitoid, Co.chilonis. These results will provide a foundation for the establishment of a new safety evaluation system for novel RNAi-based transgenic plants.

Acute Effects of Dietary Protein Consumption on the Postprandial Metabolic Response, Amino Acid Levels and Circulating MicroRNAs in Patients with Obesity and Insulin Resistance.

The post-nutritional intervention modulation of miRNA expression has been previously investigated; however, post-acute dietary-ingestion-related miRNA expression dynamics in individuals with obesity and insulin resistance (IR) are unknown. We aimed to determine the acute effects of protein ingestion from different dietary sources on the postprandial metabolic response, amino acid levels, and circulating miRNA expression in adults with obesity and IR. This clinical trial included adults with obesity and IR who consumed (1) animal-source protein (AP; calcium caseinate) or (2) vegetable-source protein (VP; soy protein isolate). Glycaemic, insulinaemic, and glucagon responses, amino acid levels, and exosomal microRNAs isolated from plasma were analysed. Post-AP ingestion, the area under the curve (AUC) of insulin (p = 0.04) and the plasma concentrations of branched-chain (p = 0.007) and gluconeogenic (p = 0.01) amino acids increased. The effects of different types of proteins on the concentration of miRNAs were evaluated by measuring their plasma circulating levels. Compared with the baseline, the AP group presented increased circulating levels of miR-27a-3p, miR-29b-3p, and miR-122-5p (p < 0.05). Subsequent analysis over time at 0, 30, and 60 min revealed the same pattern and differences between treatments. We demonstrated that a single dose of dietary protein has acute effects on hormonal and metabolic regulation and increases exosomal miRNA expression in individuals with obesity and IR.

Preanalytical considerations in quantifying circulating miRNAs that predict end-stage kidney disease in diabetes.

Our previous study identified 8 risk and 9 protective plasma miRNAs associated with progression to end-stage kidney disease (ESKD) in diabetes. This study aimed to elucidate preanalytical factors that influence the quantification of circulating miRNAs. Using the EdgeSeq platform, which quantifies 2,002 miRNAs in plasma, including ESKD-associated miRNAs, we compared miRNA profiles in whole plasma versus miRNA profiles in RNA extracted from the same plasma specimens. Less than half of the miRNAs were detected in standard RNA extraction from plasma. Detection of individual and concentrations of miRNAs were much lower when RNA extracted from plasma was quantified by RNA sequencing (RNA-Seq) or quantitative reverse transcription PCR (qRT-PCR) platforms compared with EdgeSeq. Plasma profiles of miRNAs determined by the EdgeSeq platform had excellent reproducibility in assessment and had no variation with age, sex, hemoglobin A1c, BMI, and cryostorage time. The risk ESKD-associated miRNAs were detected and measured accurately only in whole plasma and using the EdgeSeq platform. Protective ESKD-associated miRNAs were detected by all platforms except qRT-PCR; however, correlations among concentrations obtained with different platforms were weak or nonexistent. In conclusion, preanalytical factors have a profound effect on detection and quantification of circulating miRNAs in ESKD in diabetes. Quantification of miRNAs in whole plasma and using the EdgeSeq platform may be the preferable method to study profiles of circulating cell-free miRNAs associated with ESKD and possibly other diseases.

CsPbBr3 Perovskite Quantum Dots Encapsulated by a Polymer Matrix for Ultrasensitive Dynamic Imaging of Intracellular MicroRNA.

Herein, CsPbBr3 perovskite quantum dots (CPB PQDs)@poly(methyl methacrylate) (PMMA) (CPB@PMMA) nanospheres were used as energy donors with high Förster resonance energy transfer (FRET) efficiency and exceptional biocompatibility for ultrasensitive dynamic imaging of tiny amounts of microRNAs in living cells. Impressively, compared with traditional homogeneous single QDs as energy donors, CPB@PMMA obtained by encapsulating numerous CPB PQDs into PMMA as energy donors could not only significantly increase the efficiency of FRET via improving the local concentration of CPB PQDs but also distinctly avoid the problem of cytotoxicity caused by divulged heavy metal ions entering living cells. Most importantly, in the presence of target miRNA-21, DNA dendrimer-like nanostructures labeled with 6-carboxy-tetramethylrhodamine (TAMRA) were generated by the exposed tether interhybridization of the Y-shape structure, which could wrap around the surface of CPB@PMMA nanospheres to remarkably bridge the distance of FRET and increase the opportunity for effective energy transfer, resulting in excellent precision and accuracy for ultrasensitive and dynamic imaging of miRNAs. As proof of concept, the proposed strategy exhibited ultrahigh sensitivity with a detection limit of 45.3 aM and distinctly distinguished drug-irritative miRNA concentration abnormalities with living cells. Hence, the proposed enzyme-free CPB@PMMA biosensor provides convincing evidence for supplying accurate information, which could be expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

DNA-Functionalized Gold Nanorods for One-Step Simultaneous Determination of Multiple microRNAs Based upon Plasma-Enhanced Fluorescence

MicroRNAs (miRNAs) are endogenous, noncoding, and sequence-specific single-stranded RNA regulatory factors whose abnormal expression levels are closely related to various diseases, especially cancer, and are considered effective biomarkers for disease diagnosis and prognosis. Since the concentrations of miRNAs in the body are low and disease diagnosis by detecting single miRNAs may result in a high false positive, it is essential to construct a sensitive biosensor for the determination of multiple miRNAs. In this work, an enzyme-free signal amplification assay based on plasma-enhanced fluorescence (PEF) is reported for the one-step simultaneous determination of two miRNAs. Gold nanorods (AuNRs) were functionalized with fluorescein isothiocyanate (FAM)-tagged hairpin DNA1 (H1) and cyanine 5.5 (Cy5.5)-tagged hairpin DNA2 (H2). The fluorescence signals of FAM and Cy5.5 were quenched due to their proximity to the surface of the AuNRs. In the presence of target miRNA-21 and miRNA-155, H1 and H2 complemented miRNA-21 and miRNA-155, respectively, and the fluorescence signals of FAM and Cy5.5 were not only restored but amplified due to the plasma-enhanced fluorescence. A suitable linear range (0.1 to 10 nM) and low limits of detection for miRNA-21 (23 pM) and miRNA-155 (1.6 pM) were achieved. The developed assay for the one-step simultaneous measurement of two miRNAs is simple, fast and sensitive, exhibiting great potential for monitoring these biomarkers.

Preanalytical considerations for clinical assays of circulating human miRNA-451a, miRNA-423-5p and miRNA-199a-3p for diagnostic purposes.

Circulating miRNA has recently emerged as important biomolecules with potential clinical values as diagnostic markers for several diseases. However, to be used as such, it is critical to accurately quantify miRNAs in the clinic. Yet, preanalytical factors that can affect an error-free quantification of these miRNAs have not been explored. This study aimed at investigating several of these preanalytical factors that may affect the accurate quantification of miRNA-451a, miRNA-423-5p and miRNA-199a-3p in human blood samples. We initially evaluated levels of these three miRNAs in red blood cells (RBCs), white blood cells (WBCs), platelets, and plasma by droplet digital PCR (ddPCR). Next, we monitored miRNA levels in whole blood or platelet rich plasma (PRP) stored at different temperatures for different time periods by ddPCR. We also investigated the effects of hemolysis on miRNA concentrations in platelet-free plasma (PFP). Our results demonstrate that more than 97% of miRNA-451a and miRNA-423-5p in the blood are localized in RBCs, with only trace amounts present in WBCs, platelets, and plasma. Highest amount of the miRNA-199a-3p is present in platelets. Hemolysis had a significant impact on both miRNA-451a and miRNA-423-5p concentrations in plasma, however miRNA-199a levels remain unaffected. Importantly, PRP stored at room temperature (RT) or 4°C showed a statistically significant decrease in miRNA-451a levels, while the other two miRNAs were increased, at days 1, 2, 3 and 7. PFP at RT caused statistically significant steady decline in miRNA-451a and miRNA-423-5p, observed at 12, 24, 36, 48 and 72 hours. Levels of the miRNA-199a-3p in PFP was stable during first 72 hours at RT. PFP stored at -20°C for 7 days showed declining stability of miRNA-451a over time. However, at -80°C miRNA-451a levels were stable up to 7 days. Together, our data indicate that hemolysis and blood storage at RT, 4°C and -20°C may have significant negative effects on the accuracy of circulating miRNA-451a and miRNA-423-5p quantification.

Organic small molecule catalytic PET-RAFT electrochemical signal amplification strategy for miRNA-21 detection

Quantitative detection of the cancer-associated biomarker miRNA-21 is essential for the prevention and treatment of early-stage cancer patients. In the early diagnosis of cancer, due to the low concentration of miRNA in the organism, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification techniques. Herein, a photo-induced electron/energy transfer reversible addition-fragmentation chain transfer polymerization signal amplification electrochemical biosensor was developed. Peptide nucleic acid was used as the recognition probe for miRNA-21 and the chain transfer agent (4-cyano-4-(dodecylsulfonylsulfanylthiocarbonyl)sulfonylpentanoic acid) bearing a carboxylic acid group was successfully conjugated with a phosphate group in the presence of ZrOCl2 to form the phosphate-Zr(IV)-carboxylic acid complex. Subsequently, the polymerization of the monomer (ferrocenylmethyl methacrylate) was achieved by blue light irradiation in the presence of erythrosin B (higher triple quantum yield) and triethanolamine (increases electron transfer rate). With this sophisticated signal amplification biosensing platform, low detection limit of 12.4 aM was achieved for miRNA-21. The biosensor reduces costs and simplifies experimentation while ensuring high sensitivity and selectivity. Meanwhile, the biosensor exhibits excellent anti-interference ability in serum samples and has great potential for practical applications in the early prevention of cancer.

Plasma miR-203a-3p as a Novel Predictor of Dementia in Patients with Parkinson's Disease.

The early detection of cognitive decline in Parkinson's disease is important for providing drug therapy and non-pharmacological management. The circulating microRNAs present in plasma are promising biomarkers of PD with dementia (PDD) due to their critical roles in synaptic plasticity and the regulation of neurodegeneration-associated proteins. In this study, we aimed to identify plasma microRNAs that may differentiate PD with or without cognitive impairment. Global microRNA expression was obtained from a discovery set of 123 participants who were divided into four groups, namely normal controls (HC), PD with no dementia (PDND), PD with mild cognitive impairment (PD-MCI), and PDD, using next-generation sequencing. The BOLD selector was used for microRNA candidate selection. Six miRNAs, namely miR-203a-3p, miR-626, miR-662, miR-3182, miR-4274, and miR-4295, were clustered as potential candidates for use in identifying PDND from PD-MCI. Another independent cohort of 120 participants was further recruited in a validation step in order to detect candidate microRNAs via droplet digital PCR (ddPCR), which was used for its high sensitivity in detecting low miRNA concentrations. Our results show that the ratio of miR-203a-3p/miR-16-5p, in which miR-16-5p was used as a reference control miRNA, was significantly increased in PDD compared to that seen in PD-MCI and PDND individually, and was negatively correlated with the MoCA scores (r = -0.237, p = 0.024) in patients with PD. However, there was no significant difference in the ratio of miR-203a-3p/miR-16-5p between HC and PDND, PD-MCI, or PDD individually. The ROC curve of the logistic regression model, factoring in the variables of age, the ratio of miR-203a-3p/miR-16-5p, and the UPDRS III score, demonstrated an AUC of 0.883. Our findings suggest that the ratio of miR-203a-3p/miR-16-5p, used with age and motor score, could be a predictor of dementia among PD patients.

MicroRNAs as biomarkers of brain injury in neonatal encephalopathy: an observational cohort study

Neonatal Encephalopathy (NE) is a major cause of lifelong disability and neurological complications in affected infants. Identifying novel diagnostic biomarkers in this population may assist in predicting MRI injury and differentiate neonates with NE from those with low-cord pH or healthy neonates and may help clinicians make real-time decisions. To compare the microRNA (miRNA) profiles between neonates with NE, healthy controls, and neonates with low cord pH. Moreover, miRNA concentrations were compared to brain injury severity in neonates with NE. This is a retrospective analysis of miRNA profiles from select samples in the biorepository and data registry at the University of Florida Health Gainesville. The Firefly miRNA assay was used to screen a total of 65 neurological miRNA targets in neonates with NE (n = 36), low cord pH (n = 18) and healthy controls (n = 37). Multivariate statistical techniques, including principal component analysis and orthogonal partial least squares discriminant analysis, and miRNA Enrichment Analysis and Annotation were used to identify miRNA markers and their pathobiological relevance. A set of 10 highly influential miRNAs were identified, which were significantly upregulated in the NE group compared to healthy controls. Of these, miR-323a-3p and mir-30e-5p displayed the highest fold change in expression levels. Moreover, miR-34c-5p, miR-491-5p, and miR-346 were significantly higher in the NE group compared to the low cord pH group. Furthermore, several miRNAs were identified that can differentiate between no/mild and moderate/severe injury in the NE group as measured by MRI. MiRNAs represent promising diagnostic and prognostic tools for improving the management of NE.

Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA.

As an important component of highly heterogeneous exosomes, exosomal microRNAs (miRNAs) have great potential as noninvasive biomarkers for cancer diagnosis. Therefore, a sensitive and simple sensor is the key for its clinical application. Herein, we designed an exponential amplification reaction (EXPAR) to induce the reactivation of the CRISPR-associated protein 9/small guide RNA (Cas9/sgRNA) complex, thus achieving sensitive and visual exosomal miRNAs-21 (miR-21) fluorescence sensing. In this design, we inactivated the sgRNA by hybridizing sgRNA and blocker DNA. Then, we used a trigger DNA to hybridize with miR-21 and produced a lot of activated DNA by EXPAR. Those activated DNA further hybridized with blocker DNA and released the free sgRNA to form the activated Cas9/sgRNA complex. Based on the quick cleavage of activated Cas9/sgRNA complex, the reporter DNA labeled by SYBR Green I was released from the surface of the magnetic nanoparticles (MNPs) into the supernatant, and thus was used to sensitively quantify the miRNAs concentration with a limit of detection of 3 × 103 particles/mL. In addition, this fluorescence sensor has also been successfully employed to distinguish healthy people and cancer patients by naked-eye observation of the fluorescence, thus demonstrating its great potential for accurate and point-of-care cancer diagnosis.

Evaluation of New Cardiac Damage Biomarkers in Polytrauma: GDF-15, HFABP and uPAR for Predicting Patient Outcomes.

Background: Polytrauma is one of the leading mortality factors in younger patients, and in particular, the presence of cardiac damage correlates with a poor prognosis. Currently, troponin T is the gold standard, although troponin is limited as a biomarker. Therefore, there is a need for new biomarkers of cardiac damage early after trauma. Methods: Polytraumatized patients (ISS ≥ 16) were divided into two groups: those with cardiac damage (troponin T > 50 pg/mL, n = 37) and those without cardiac damage (troponin T < 12 pg/mL, n = 32) on admission to the hospital. Patients' plasma was collected in the emergency room 24 h after trauma, and plasma from healthy volunteers (n = 10) was sampled. The plasma was analyzed for the expression of HFABP, GDF-15 and uPAR proteins, as well as miR-21, miR-29, miR-34, miR-122, miR-125b, miR-133, miR-194, miR-204, and miR-155. Results were correlated with patients' outcomes. Results: HFABP, uPAR, and GDF-15 were increased in polytraumatized patients with cardiac damage (p < 0.001) with a need for catecholamines. HFABP was increased in non-survivors. Analysis of systemic miRNA concentrations showed a significant increase in miR-133 (p < 0.01) and miR-21 (p < 0.05) in patients with cardiac damage. Conclusion: All tested plasma proteins, miR-133, and miR-21 were found to reflect the cardiac damage in polytrauma patients. GDF-15 and HFABP were shown to strongly correlate with patients' outcomes.