miRNA Concentrations Research Articles

Simultaneous Detection of Exosomal microRNAs Isolated from Cancer Cells Using Surface Acoustic Wave Sensor Array with High Sensitivity and Reproducibility.

We present a surface acoustic wave (SAW) sensor array for microRNA (miRNA) detection that utilizes photocatalytic silver staining on titanium dioxide (TiO2) nanoparticles as a signal enhancement technique for high sensitivity with an internal reference sensor for high reproducibility. A sandwich hybridization was performed on working sensors of the SAW sensor array that could simultaneously capture and detect three miRNAs (miRNA-21, miRNA-106b, and miRNA-155) known to be upregulated in cancer. Sensor responses due to signal amplification varied depending on the concentration of synthetic miRNAs. It was confirmed that normalization (a ratio of working sensor response to reference sensor response) screened out background interferences by manipulating data and minimized non-uniformity in the photocatalytic silver staining step by suppressing disturbances to both working sensor signal and reference sensor signal. Finally, we were able to successfully detect target miRNAs in cancer cell-derived exosomal miRNAs with performance comparable to the detection of synthetic miRNAs.

P064 Investigating the genotoxic effect of reduced glutathione, n-acetylcysteine, human umbilical cord-derived mesenchymal stem cell exosomes and let-7 miRNA mimics in human lymphocytes and EpiIntestinal cells

Abstract Background The current treatments for IBD frequently do not provide sufficient control over the disease, warranting the investigation of alternative therapeutic options with minimal risk of side effects. In IBD, the overgeneration and insufficient removal of reactive oxygen species (ROS) leads to oxidative stress. Reduced glutathione (GSH) and n-acetylcysteine (NAC) are antioxidants which may possess therapeutic potential in IBD, by modulating endogenous mechanisms that decrease ROS production or increase antioxidant enzymes. Additonally, exosomes from human umbilical cord blood derived mesenchymal stem cells (hucMSCs-exo) may be used to treat IBD. These are a subset of extracellular nanosized membrane vesicles which participate in intercellular communication by delivering their contents, such as functional miRNAs to recipient cells, thereby influencing the physiological and pathological processes in various diseases. The therapeutic potential of exosomes can be enhanced through the overexpression of the let-7 family of miRNAs. Methods The EpiIntestinal tissue model is a physiologically relevant predictor of drug-induced GI toxicity. EpiIntestinal cells and lymphocytes from 3 IBD patients were transfected with 50 μL miRNA mimics and inhibitors, and treated with 50μL hucMSC-exo, and 50 μL 1mM GSH and NAC. Next, in comparison to negative controls, single-stranded DNA damage was assessed in peripheral blood mononuclear cells (PBMCs) from IBD patients using the fast microplate DNA damage assay, and double-stranded DNA damage was assessed in EpiIntestinal cells using the alkaline Comet assay. In addition, the viability of healthy and IBD lymphocytes was assessed following treatment with selected concentrations of hucMSC-exo, GSH and NAC. Results Double-stranded DNA damage was significantly reduced in EpiIntestinal cells compared to the negative control following treatment with hucMSC-exo, GSH, NAC, and miRNA mimics for let-7a-5p, -7b-5p, -7c-3p, -7d-3p and -7d-5p. In addition, single-stranded DNA damage was reduced in IBD PBMCs compared to the negative control following treatment with hucMSC-exo, and let-7b-5p and -7c-3p mimics, but was increased following treatment with inhibitors for let-7b-5p, -7c-5p and two novel miRNAs. Additionally, GSH, NAC and hucMSC-exo increased cell viability in treated versus untreated lymphocytes from healthy individuals and IBD patients. Conclusion Selected concentrations of HucMSC-exo, GSH, NAC and/or let-7 miRNA mimics reduced DNA damage and increased cell viability in treated versus untreated cells which may reduce the risk of colorectal cancer in IBD patients. Acknowledgements Our gratitude goes to the MatTek Team for generously granting us a SMI-100-FT EpiIntestinal kit in support of this research.

Integration of catalytic hairpin assembly probes into microneedles for detection of MicroRNA in plants

MicroRNAs (miRNAs) are crucial in regulating plant growth and their response to abiotic stress. Traditional miRNA detection methods are techniques which are often criticized for being expensive, labor-intensive, and time-consuming. In our research, we present a novel method that combines absorbent microneedles with a catalytic hairpin assembly (CHA) detection system. This integration facilitates swift plant sap extraction and miRNA detection. The CHA system, which consists of two hairpin probes, showcases exceptional sensitivity in identifying target miRNAs, boasting a notable detection limit of 89 pM. By modulating the photo-crosslinking duration, we controlled the swelling attributes of the methacrylic acid-modified hyaluronic acid microneedles. These microneedles proved efficient in rapidly absorbing solutions and maintained sturdy mechanical properties, allowing easy penetration into tobacco leaves for plant sap absorption. Additionally, after absorbing the miRNA solution, the fluorescence intensity of the integrated microneedles correlated linearly with concentration. It's noteworthy that while the microneedles responded to miRNA within plant tissues, it was predominantly at elevated miRNA concentrations. Our findings underscore the potential of hydrophilic microneedle arrays in both extracting and detecting miRNA in plant leaves, emphasizing their promising role in the rapid identification of vital plant compounds. This innovative method paves the way for groundbreaking research and analysis in plant studies.

Assessment of tear derived extracellular vesicles as a minimally source of novel biomarkers for subconjunctival glaucoma surgery

Aims/purpose: Glaucoma is a highly prevalent pathology causing avoidable blindness. Topical treatments used to stabilize intraocular pressure (IOP) may produce several side effects including inflammatory reactions. Patients who fail to control IOP or suffer significant side effects undergo subconjunctival glaucoma surgery (SGS). Estimations point to 30–40% of these patients will develop local scarring and fibrosis, which are the main complications of SGS. Thus, predicting the surgical outcome and starting preemptively fibrosis treatment are crucial. The aim of this study was to determine whether tears, and specifically tear‐derived extracellular vesicles (L‐EVs), can be used as liquid biopsy source, as a previous step for identifying new biomarkers for SGS outcome.Methods: Tear samples were obtained from patients (no surgical treatment, n = 11) by Schirmer's test. L‐EVs were enriched by size‐exclusion chromatography. Protein quantification was measured by microBCA assay and miRNA extraction (both from whole tear and L‐EVs) was performed using commercial kits.Results: Whole tear samples displayed variable miRNA and protein concentrations among patients (range 222.0–1388.1 ng miRNAs and 5.6–27.2 μg protein). Levels of miRNA and protein were also highly variable but considerably reduced in L‐EVS (0.14–46.78 ng miRNAs and 0.5–4.2 μg protein). Yet, in most patients, both types of samples (whole tears and L‐EVs) rendered sufficient quantity of protein and miRNA to perform proteomic and transcriptomic analysis.Conclusions: Concentration of miRNAs and Protein in tears and L‐EVs is highly variable among patients. As expected by their higher enrichment, miRNA and protein concentrations in L‐EVs are reduced compared to whole tears, yet quantities may be still enough to perform mic analyses. This higher purification of L‐EVS may help identifying specific biomarkers related to SGS outcome in a minimally invasive manner.

Decomposition Mueller matrix polarimetry for enhanced miRNA detection with antimonene-based surface plasmon resonance sensor and DNA-linked gold nanoparticle signal amplification

A decomposition Mueller matrix method is proposed for detection of miRNA and enhanced by using a surface plasmon resonance (SPR). In the proposed approach, a Mueller matrix decomposition method is employed to extract the linear birefringence (LB) and circular dichroism (CD) properties of the miRNA sample. The accuracy of the LB and CD measurements is enhanced through the use of a high-resolution antimonene-based SPR prism coupler with DNA-linked gold nanoparticles (AuNPs). The feasibility of the proposed method is demonstrated by measuring the LB orientation angle (α) and CD property (R) of two miRNA aqueous solutions (hsa-miR-125-5p and hsa-miR-21-5p) over the concentration range of 0∼1000 fM in both cases. The results show that, for both samples, α and R vary linearly with the change in the miRNA concentration. Furthermore, the values of α and R obtained for the two samples are quantifiably different, and hence the selectivity of the proposed SPR sensor is confirmed. Overall, the results highlight the potential of the proposed sensor as a valuable tool for miRNA detection with prospective applications in cancer diagnosis.

Resveratrol modulates miRNA machinery proteins in different types of colon cancer cells

Abstract Objectives Resveratrol (RSV) is a stilbenoid compound that shows anticancer activity in many cancer cells. Exosomes might affect carcinogenesis and the development of colorectal cancer by affecting communication between tumor cells in the tumor microenvironment via their cargo content miRNA. The aim of this study is to determine the effect of RSV on the expression of Dicer, Ago2, eIf2α, CD-9, CD-63, and exosomal miRNA levels in COLO320 and COLO741 colon cancer cell lines. Methods The MTT method was used for cell growth and cytotoxicity in both COLO320 and COLO741 cell lines. Dicer, Ago2, eIF2α, CD-9, and CD-63 antibodies were used for the immunocytochemical evaluation. Total miRNA analysis was performed using a miRCURY Exosome Isolation Kit. Results As a result of immunocytochemical staining, increased CD-63 immunoreactivity was observed in RSV-treated COLO320 cells vs. RSV-treated COLO-741 cells. Dicer immunoreactivity increased after the RSV treatment in COLO320 cells. Higher eIF2α immunoreactivity was observed in RSV-treated COLO741 cells compared to both COLO741 control cells and RSV-treated COLO320 cells. Non-significant decreases were observed in miRNA concentration in RSV-treated COLO320 and COLO741 cells compared to control group cells. Conclusions RSV could increase miRNA biogenesis in COLO320 cancer cells and decrease it in COLO741 cancer cells.

Exploring Optimal Biomarker Sources: A Comparative Analysis of Exosomes and Whole Plasma in Fasting and Non-Fasting Conditions for Liquid Biopsy Applications.

The study of liquid biopsy with plasma samples is being conducted to identify biomarkers for clinical use. Exosomes, containing nucleic acids and metabolites, have emerged as possible sources for biomarkers. To evaluate the effectiveness of exosomes over plasma, we analyzed the small non-coding RNAs (sncRNAs) and metabolites extracted from exosomes in comparison to those directly extracted from whole plasma under both fasting and non-fasting conditions. We found that sncRNA profiles were not affected by fasting in either exosome or plasma samples. Our results showed that exosomal sncRNAs were found to have more consistent profiles. The plasma miRNA profiles contained high concentrations of cell-derived miRNAs that were likely due to hemolysis. We determined that certain metabolites in whole plasma exhibited noteworthy concentration shifts in relation to fasting status, while others did not. Here, we propose that (1) fasting is not required for a liquid biopsy study that involves both sncRNA and metabolomic profiling, as long as metabolites that are not influenced by fasting status are selected, and (2) the utilization of exosomal RNAs promotes robust and consistent findings in plasma samples, mitigating the impact of batch effects derived from hemolysis. These findings advance the optimization of liquid biopsy methodologies for clinical applications.

Physical and physiological characteristics, as well as miRNA concentrations, are affected by the storage time of tomatoes.

This study aims to investigate the potential of miRNA measurements in indicating tomato quality during transportation and storage. The impact of storage temperature, duration, and mechanical loading on tomato senescence, carotenoid content, total soluble solids, fruit firmness, and relevant miRNA concentrations were examined. Significant two-way or three-way interactions were observed between storage conditions and physical/physiological characteristics (excluding carotenoids). Remarkably, significant three-way interactions were found between storage conditions and miRNA concentrations. Strong correlations were observed between the physiological characteristics of the tomatoes and their miRNA concentrations. These findings suggest that measuring miRNAs could serve as a convenient and portable method for evaluating postharvest fruit quality, reducing reliance on labor-intensive laboratory techniques.

Highly sensitive electrochemical assay based on strand displacement polymerization-assisted CRISPR/Cas12a collateral cleavage

In this work, a novel strand displacement polymerization strategy is designed with a single hairpin-structured DNA probe acting as both of primer and template. After target miRNA-induced structural transitions, extension and displacement reactions occur at the dimer with recycled target miRNA. The generated double-stranded products further activate CRISPR/Cas12a collateral cleavage of substrate DNA at the electrode surface, which can be reflected by the silver stripping peak variations. By analyzing the reduced peak intensity, the concentration of miRNA can be determined. This method combines the strand displacement amplification and CRISPR/Cas12a’s shearing capability. Thus a highly sensitive electrochemical biosensor is established with the limit of detection as low as 31 aM. It may also inspire new ideas of electrochemical platforms integrating CRISPR/Cas system for biological studies and clinical applications.

Urinary-derived extracellular vesicle microRNAs as non‐invasive diagnostic biomarkers for early-stage renal cell carcinoma

Background and aimsThe potential of urinary-derived extracellular vesicle (uEV) microRNAs (miRNAs) as noninvasive molecular biomarkers for identifying early-stage renal cell carcinoma (RCC) patients is rarely explored. The present study aims to explore the possibility of uEV miRNAs as novel molecular biomarkers for distinguishing early-stage RCC. Materials and methodsuEVs were extracted by ExoQuick-TC™ kit and miRNA concentrations were measured by RT-qPCR. ROC curves and bioinformatics analysis were employed to predict the diagnostic efficacy and regulatory mechanisms of dysregulated miRNAs. ResultsThrough a multiphase case-control study on uEV miRNAs screening, training, and validation in RCC cells (ACHN, Caki-1) and control cells (HK-2) and in uEVs of 125 RCC patients and 128 age- and sex-matched controls, we successfully identified four uEVs miRNAs (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) were significantly and stably upregulated in RCC in vitro and in vivo. When adjusted with estimated glomerular filtration rate (eGFR), the AUC of the three-uEV miRNA panel (miR-135b-5p, miR-200c-3p, and miR-203a-3p) was 0.785 (95 % CI = 0.729–0.842, P < 0.0001) for discriminating RCC patients from controls. Notably, this panel exhibited similar performance in distinguishing early-stage (stage Ⅰ) RCC patients, with an AUC of 0.786 (95 %CI = 0.727–0.844, P < 0.0001). Bioinformatics analysis predicted that candidate miRNAs were involved in cancer progressing. ConclusionOur study identified a four uEV miRNAs panel (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) may serve as an auxiliary noninvasive indication of early-stage RCC.

The Role of miRNA in Testicular Cancer: Current Insights and Future Perspectives.

Background and Objectives: Despite advancements in the diagnosis and treatment of testicular germ cell tumours (TGTCs), challenges persist in identifying reliable biomarkers for early detection and precise disease management. This narrative review addresses the role of microRNAs (miRNAs) as potential diagnostic tools and therapeutic targets in the treatment of TGCTs. Materials and Methods: Three databases (PubMed®, Web of Science™, and Scopus®) were queried for studies investigating the utility of miRNA as diagnostic tools, assessing their prognostic significance, and evaluating their potential to guide TGCT treatment. Different combinations of the following keywords were used, according to a free-text protocol: "miRNA", "non-coding RNA", "small RNA", "Testicular Cancer", "seminomatous testicular germ cell", "non-seminomatous testicular germ cell". Results: The potential of miRNAs as possible biomarkers for a non-invasive diagnosis of TGCT is appealing. Their integration into the diagnostic pathway for TGCT patients holds the potential to enhance the discriminative power of conventional serum tumour markers (STMs) and could expedite early diagnosis, given that miRNA overexpression was observed in 50% of GCNIS cases. Among miRNAs, miR-371a-3p stands out with the most promising evidence, suggesting its relevance in the primary diagnosis of TGCT, particularly when conventional STMs offer limited value. Indeed, it demonstrated high specificity (90-99%) and sensitivity (84-89%), with good positive predictive value (97.2%) and negative predictive value (82.7%). Furthermore, a direct relationship between miRNA concentration, disease burden, and treatment response exists, regardless of disease stages. The initial evidence of miRNA decrease in response to surgical treatment and systemic chemotherapy has been further supported by more recent results suggesting the potential utility of this tool not only in evaluating treatment response but also in monitoring residual disease and predicting disease relapse. Conclusions: MiRNAs could represent a reliable tool for accurate diagnosis and disease monitoring in the treatment of TGCT, providing more precise tools for early detection and treatment stratification. Nevertheless, well-designed clinical trials and comprehensive long-term data are needed to ensure their translation into effective clinical tools.

Hybridization chain reaction assisted terahertz metamaterial biosensor for highly sensitive detection of microRNAs

MicroRNA (miRNA) is closely related to the occurrence and development of cancer. Accurate determination of the miRNA concentration is of great significance for early cancer diagnosis. However, due to the short sequence and low concentration of miRNA, it is still a challenge to achieve low-concentration detection. In this work, we proposed a method for the highly sensitive detection of miRNA-21 using a terahertz (THz) metamaterial sensor combined with a Hybridization chain reaction (HCR). First, a capture hairpin probe was combined with gold nanoparticles (AuNPs), which were then modified to the surface of the sensor for specific binding of miRNA-21. Then the signal amplification technique of HCR is used to amplify the trace amount of miRNA, and the super-long dendritic DNA macromolecules are formed on the surface of the sensor. This changes the dielectric environment of the sensor surface, and the resonance frequency of the sensor is shifted. The method has good specificity and sensitivity, and the concentration of miRNA-21 in the range of 100 aM to 10 nM shows excellent linear relationship with frequency shift. Most importantly, it paves the way for low-cost, easy-to-operate and marker-free miRNA detection.

Electrochemical Biosensor for Ultrasensitive Detection of Microrna‐21 through In Situ Synthesis of Palladium Nanoparticles

AbstractThe detection of trace microRNAs (miRNA) is crucial for early cancer diagnosis. Here, a sensitive biosensor for miRNA directive detection was put forward, which was based on RNA‐templated palladium nanoparticles (PdNPs) signal amplification strategy. Uncharged peptide nucleic acids (PNA) probe was used to capture the target miRNA(T‐RNA) specifically. Subsequently, a large amount of palladium ions absorbed on T‐RNA strands via electrostatic forces, and the palladium nanocluster is rapidly generated based on chemical reduction. Obviously, RNA metallization method enables biosensor to be carried out efficiently at room temperature also simplifies the preparation and detection of the sensor. Under optimal conditions, this method can analyze the concentration of miRNA in the range of 10 fM to 1 nM, and the detection limit is 3.33 fM. At the same time, this method has strong anti‐interference ability in serum samples, which provides a broad prospect for the early screening and diagnosis of cancer.

Machine learning provides specific detection of salt and drought stresses in cucumber based on miRNA characteristics

BackgroundSpecific detection of the type and severity of plant abiotic stresses helps prevent yield loss by considering timely actions. This study introduces a novel method to detect the type and severity of stress in cucumber plants under salinity and drought conditions. Various features, i.e., morphological (image textural features), physiological/biochemical (relative water content, chlorophyll, catalase activity, anthocyanins, phenol content, and proline), as well as miRNA characteristics (the concentration of miRNA-156a, miRNA-166i, miRNA-399g, and miRNA-477b) were extracted from plant leaves, and machine learning methods were used to predict the type and severity of stress by having these features. Support vector machine (SVM) with parameters optimized by genetic algorithm (GA) and particle swarm optimization (PSO) was used for machine learning.ResultsThe coefficient of determination of predicting the stress type and severity in plants under both stresses was 0.61, 0.82, and 0.99 using morphological, physiological/biochemical, and miRNA characteristics, respectively. This reveals machine learning methods optimized by metaheuristic optimization techniques can provide specific detection of salt and drought stresses in cucumber plants based on miRNA characteristics. Among the study miRNAs, miRNA-477b and miRNA-399g had the highest and lowest contribution to salt and drought stresses, respectively.ConclusionsComapred to conventional plant traits, miRNAs are more reliable features for providing us with valuable information about plant abiotic diseases at early stages. Using an electrochemical miRNA biosensor similar to one used in this work to measure the miRNA concentration in plant leaves and using a machine learning algorithm such as SVM enable farmers to detect the salt and drought stress at early stages in cucumber plants with very high accuracy.

Quercetin Change the Exosome Secretion and Total miRNA Concentration in Primary (Colo320) and Metastatic (Colo741) Colon Cancer Cell Lines

Quercetin Change the Exosome Secretion and Total miRNA Concentration in Primary (Colo320) and Metastatic (Colo741) Colon Cancer Cell Lines

Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk.

Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 1010 (±3.30 × 109) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%-16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development.

Isolation and Detection of Exosomal Mir210 Using Carbon Nanomaterial-Coated Magnetic Beads.

MicroRNAs (miRNAs) are short non-coding RNAs that are found in various cellular compartments and play an important role in regulating gene expression. Extracellular miRNAs, such as those found within extracellular vesicles such as exosomes are involved in cell-to-cell communication. The intercellular transfer of miRNAs has been implicated in various diseases' pathogenesis including cancer and has been studied extensively as potential cancer biomarkers. However, the extraction of miRNA from exosomes is still a challenging task. The current nucleic acid extraction assays are expensive and labor-intensive. In this study, we demonstrated a microfluidic device for aptamer-based magnetic separation of the exosomes and subsequent detection of the miRNA using a fluorescence switching assay, which was enabled by carbon nanomaterials coated on magnetic beads. In the OFF state, the fluorophore-labelled cDNA is quenched using carbon nanomaterials. However, when the target miRNA210 is introduced, the cDNA detaches from the bead's surface, which leads to an increase in the fluorescence intensity (ON state). This increment was found to be proportional to miRNA concentration within the dynamic range of 0-100 nM with a detection limit of 5 pM. The assay was validated with spiked miRNA using the standard RT-PCR method. No notable cross-reactivity with other closely related miRNAs was observed. The developed method can be utilized for the minimally invasive detection of cancer biomarkers.

A simple and sensitive turn-off biosensor for miRNA-21 detection based on tyramine signal amplification

The expression level of microRNA (miRNA) is strongly associated with a variety of diseases, and the development of new approaches for miRNA assay with high sensitivity will facilitate a deeper understanding of miRNA biological functions. Herein, a simple and sensitive turn-off biosensor for miRNA-21 detection based on tyramine signal amplification (TSA) was developed. In the absence of the target miRNA, the DNA capture probe on the surface of magnetic bead (MB) complementarily hybridizes with the DNA reporter probe; subsequently, a large amount of horseradish peroxidase (HRP) is bound by the biotin-streptavidin reaction; later, in the presence of hydrogen peroxide (H2O2), HRP catalyzes the deposition of biotinylated tyramine, and then the fluorescent phycoerythrin (PE) is successfully introduced via a biotin-streptavidin reaction, producing a robust fluorescence (FL) signal upon excitation. When the target miRNA is present, it competitively occupies the binding site of the DNA capture probes, leading to a decrease in the amount of the DNA reporter probes hybridized to the MB surface; subsequently, the effect of TSA is diminished, which ultimately leads to a weakened FL and generates the “turn-off” signal. This method utilizes the negative correlation between target miRNA concentration and FL intensity, which can achieve high sensitivity detection of miRNA with low detection limit. This method can be successfully applied to the determination of miRNA-21 in human lung cancer cells, which has great application potential for disease diagnosis.

Effects of Cow's Milk Processing on MicroRNA Levels.

MicroRNAs (miRNAs) regulate gene expression and might resist adverse physicochemical conditions, which makes them potential biomarkers. They are being investigated as biomarkers of dairy production systems, based on the variations in their levels in raw milk depending on animal diet and management. Whether miRNA levels can serve as biomarkers for dairy products remains unclear, since technological or culinary treatments, such as fermentation, may alter their levels. Here, 10 cow dairy farms were sampled in Asturias (north-west Spain) and milk samples were subjected to microwave heating or used to produce yogurt or cheese. Total RNA was isolated from raw milk and three derived products, and levels of seven miRNAs, selected based on previous studies as possible milk production system biomarkers, were assessed by RT-qPCR. The treatments decreased levels of all miRNAs to some extent. These results also imply that cheesemaking increases the concentration of miRNAs in this product; raw milk and cheese supposedly may provide similar concentrations of miRNAs, higher than those of yogurt and microwaved milk. They also indicate that the content of certain miRNAs in raw milk cannot necessarily be extrapolated to other dairy products.

MiRNA 511_5p is a potential biomarker for ocular toxoplasmosis.

Ocular toxoplasmosis (OT) is a frequent clinical manifestation due to infection by Toxoplasma gondii. It is characterized by an inflammatory process involving macrophages activated by pro-inflammatory cytokines. The expression of microRNAs takes place during the inflammatory process and, among them, miRNA 511 regulates the activation of macrophages. This study evaluated the expression of miRNA 511_5p in patients with OT and healthy controls. A total of 361 patients from the Hospital de Base of Fundação Faculdade de Medicina de São José do Rio Preto were enrolled and divided into four groups: G1-patients with active ocular lesions and reagent serology for T. gondii; G2-patients with scars and reagent serology for T. gondii; G3-patients without ocular lesions or scars and reagent serology for T. gondii; G4-patients without ocular lesions or scars and non-reagent serology for T. gondii. All patients underwent clinical and laboratory evaluation to confirm the diagnosis of OT. Serology tests, RNA extraction and cDNA synthesis were performed. The miRNA 511_5p levels were compared among the groups. The G1 group showed a high blood plasma concentration of miRNA 511_5p (mean 22.34) compared with the G2 (4.65), G3 (8.91) and G4 (3.52) groups (p<0.0001). These data suggest that miRNA 511_5p has significant potential as a biomarker for OT.