Imaging Flow Cytometry Demonstrates Physiological and Morphological Diversity within Treated Probiotic Bacteria Groups.

Probiotic bacteria can be introduced to stresses during the culturing phase as an alternative to the use of protectants and coating substances during drying. Accurate enumeration of the bacterial count in a probiotic formulation can be provided using imaging flow cytometry (IFC). IFC overcomes the weak points of conventional, commonly used flow cytometry by combining its statistical power with the imaging content of microscopy in one system. Traditional flow cytometers only collect the fluorescence signal intensities, while IFC provides many more steps as it correlates the data on the measured parameters of fluorescence light with digitally processed images of the analyzed cells. As an alternative to standard methods (plate cell counts and traditional flow cytometry) IFC provides additional insight into the physiology and morphology of the cell. The use of complementary dyes (RedoxSensorTM Green and propidium iodide) allows for the designation of groups based on their metabolic activity and membrane damage. Additionally, cell sorting is incorporated to assess each group in terms of growth on different media (MRS-Agar and MRS broth). Results show that the groups with intermediate metabolic activity and some degree of cellular damage correspond with the description of viable but nonculturable cells.

Imaging Flow Cytometry to Study Biofilm-Associated Microbial Aggregates.

The aim of the research was to design an advanced analytical tool for the precise characterization of microbial aggregates from biofilms formed on food-processing surfaces. The approach combined imaging flow cytometry with a machine learning-based interpretation protocol. Biofilm samples were collected from three diagnostic points of the food-processing lines at two independent time points. The samples were investigated for the complexity of microbial aggregates and cellular metabolic activity. Thus, aggregates and singlets of biofilm-associated microbes were simultaneously examined for the percentages of active, mid-active, and nonactive (dead) cells to evaluate the physiology of the microbial cells forming the biofilm structures. The tested diagnostic points demonstrated significant differences in the complexity of microbial aggregates. The significant percentages of the bacterial aggregates were associated with the dominance of active microbial cells, e.g., 75.3% revealed for a mushroom crate. This confirmed the protective role of cellular aggregates for the survival of active microbial cells. Moreover, the approach enabled discriminating small and large aggregates of microbial cells. The developed tool provided more detailed characteristics of bacterial aggregates within a biofilm structure combined with high-throughput screening potential. The designed methodology showed the prospect of facilitating the detection of invasive biofilm forms in the food industry environment.

Flow cytometric approach to evaluate the impact of hydro-technical concrete compounds' release to the freshwater microbiome.

The aim of this research was to test the potential of applying a flow cytometric procedure to evaluate the impact of concrete compounds' release to the freshwater microbiome. Cells from the collected samples were stained with a fluorogenic redox indicator dye that measures the redox potential of microbial cells. This novel approach was combined with the assessment of microorganisms' penetration into the internal structures of concrete using the Rose Bengal sodium salt staining. Rose Bengal staining revealed an intense fouling of the upper and side walls of the concrete cubes and also indicated the penetration of microorganisms inside the concrete as observed for the cubes' cross-sections. Flow cytometric cellular redox potential measurement revealed high percentages of active cells within the concrete's porous structures and in non-exposed water (32.7% and 30.2% of active cells) versus samples from exposed water and concrete's outer surfaces (6.8%, 6.1%, and 3.3% of active cells). The results demonstrated a detrimental impact of hydro-technical concrete on the vitality of microbial cells within the freshwater environment. Tested protocol by analyzing the physiology of microbial cells improved the functional description of complex communities to evaluate the fate of contaminants present in the concrete-based hydro-technical infrastructure.

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.

Heavy Metals as a Factor Increasing the Functional Genetic Potential of Bacterial Community for Polycyclic Aromatic Hydrocarbon Biodegradation.

The bioremediation of areas contaminated with hydrocarbon compounds and heavy metals is challenging due to the synergistic toxic effects of these contaminants. On the other hand, the phenomenon of the induction of microbial secretion of exopolysaccharides (EPS) under the influence of heavy metals may contribute to affect the interaction between hydrophobic hydrocarbons and microbial cells, thus increasing the bioavailability of hydrophobic organic pollutants. The purpose of this study was to analyze the impact of heavy metals on the changes in the metapopulation structure of an environmental consortium, with particular emphasis on the number of copies of orthologous genes involved in exopolysaccharide synthesis pathways and the biodegradation of hydrocarbons. The results of the experiment confirmed that the presence of heavy metals at concentrations of 50 mg·L-1 and 150 mg·L-1 resulted in a decrease in the metabolic activity of the microbial consortium and its biodiversity. Despite this, an increase in the biological degradation rate of polycyclic aromatic hydrocarbons was noted of 17.9% and 16.9%, respectively. An assessment of the estimated number of genes crucial for EPS synthesis and biodegradation of polycyclic aromatic hydrocarbons confirmed the relationship between the activation of EPS synthesis pathways and polyaromatic hydrocarbon biodegradation pathways. It was established that microorganisms that belong to the Burkholderiales order are characterized by a high representation of the analyzed orthologs and high application potential in areas contaminated with heavy metals and hydrocarbons.

Positive cofactor 4 (PC4) contributes to the regulation of replication-dependent canonical histone gene expression.

BACKGROUND: Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this increment is achieved by both enhanced transcription and 3' end processing. In this paper, we described positive cofactor 4 (PC4) as a protein that contributes to the regulation of replication-dependent histone gene expression. RESULTS: We showed that PC4 influences RNA polymerase II recruitment to histone gene loci in a cell cycle-dependent manner. The most important effect was observed in S phase where PC4 knockdown leads to the elevated level of RNA polymerase II on histone genes, which corresponds to the increased total level of those gene transcripts. The opposite effect was caused by PC4 overexpression. Moreover, we found that PC4 has a negative effect on the unique 3' end processing of histone pre-mRNAs that can be based on the interaction of PC4 with U7 snRNP and CstF64. Interestingly, this effect does not depend on the cell cycle. CONCLUSIONS: We conclude that PC4 might repress RNA polymerase II recruitment and transcription of replication-dependent histone genes in order to maintain the very delicate balance between histone gene expression and DNA synthesis. It guards the cell from excess of histones in S phase. Moreover, PC4 might promote the interaction of cleavage and polyadenylation complex with histone pre-mRNAs, that might impede with the recruitment of histone cleavage complex. This in turn decreases the 3' end processing efficiency of histone gene transcripts.

The effect of eubiotic feed additives on the performance of growing pigs and the activity of intestinal microflora.

The aim of this study was to compare the effect of probiotic bacteria, prebiotics, phytobiotics and their combinations on performance and microbial activity in the digestive tract of growing pigs. The experiment was conducted over 28 d on 48 male pigs of about 12 kg body weight (BW), which were allocated to following treatments.: (1) Control Group (Con) without additive, (2) Group I, addition of a prebiotic (inulin), (3) Group Ph, a phytobiotic (herbal water extracts), (4) Group P, a probiotic composed of four strains of lactic acid bacteria, (5) Group PhP, phytobiotic and probiotic bacteria and (6) Group PhPI, a phytobiotic, probiotic bacteria and a prebiotic. Animal performance was recorded and at d 28 six pigs from each group were euthanised to collect digesta samples. In all groups except for Group I, diarrhoea incidents were observed. Groups Ph and P had significantly higher daily gains and final BW, and Group Ph utilised feed better than other groups. The pH of ileal digesta was significantly lower in Group PhPI. In the caecal digesta of Groups I, P and PhP, the pH level was lower than in the other groups but dry matter contents was significantly higher in Groups Con and I. The short-chain fatty acids and particular acid content differed significantly only in the colonic digesta. The yeast and mould numbers in caecal digesta was highest in Group Con. No treatment effects were observed for the number of lactic acid bacteria, coli group bacteria or Clostridium. However, the observed significantly higher number of total bacteria suggests that a multi-component eubiotic treatment changes the bacterial composition and distribution more effectively. Our findings indicated that all used additives changed the intestinal microflora, but the multi-component eubiotics were not beneficial as feed additives offered separately. Moreover, supplementation of phytobiotics and probiotic bacteria also improved the animal performance significantly.

Investigation of the effectiveness of disinfectants against planktonic and biofilm forms of P. aeruginosa and E. faecalis cells using a compilation of cultivation, microscopic and flow cytometric techniques.

This study evaluated the effectiveness of selected disinfectants against bacterial cells within a biofilm using flow cytometry, the conventional total viable count test and scanning electron microscopy (SEM). A flow cytometric procedure based on measurement of the cellular redox potential (CRP) was demonstrated to have potential for the rapid evaluation of activity against biofilm and planktonic forms of microbes. Quaternary ammonium compound-based disinfectant (QACB) demonstrated a higher level of anti-microbial activity than a performic acid preparation (PAP), with mean CRP values against P. aeruginosa cells of 2 and 1.33 relative fluorescence units (RFU) vs 63.33 and 61.33 RFU for 8 and 24 h cultures respectively. Flow cytometric evaluation of the anti-biofilm activity demonstrated a higher efficacy of QACB compared to PAP for P. aeruginosa cells of 1 and 0.66 RFU vs 18.33 and 22.66 RFU for 8 and 24 h cultures respectively. SEM images of treated P. aeruginosa cells demonstrated disinfectant-specific effects on cell morphology.

Lactobionic acid production by glucose-fructose oxidoreductase from Zymomonas mobilis expressed in Escherichia coli.

OBJECTIVES: To use the glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis and expressed in Escherichia coli for lactobionic acid production by conversion of lactose from whey. RESULTS: The highest concentrations of lactobionic acid (3.2 mg ml(-1)) during oxidation of whey-derived lactose by E. coli was at 24 h. Introduction of GFOR gene from Z. mobilis, into E. coli improved enzyme yields compared to what is obtainable by fermentation of the donor strain. The production of lactobionic acid by E. coli was 2.6-times higher than by Z. mobilis. CONCLUSIONS: Recombinants of E. coli overexpressing the GFOR gene from Z. mobilis produced higher amount of lactobionoic acid from whey-derived lactose.

Antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion.

BACKGROUND: Broccoli is a common vegetable recognized as a rich source of antioxidants. To date, research on the antioxidant properties of broccoli, predominantly conducted on extracts, has not considered the lesions of composition and this activity after gastrointestinal digestion. Here the stability of antioxidants during gastrointestinal digestion was evaluated in conjunction with the protective effects of broccoli sprouts (BS) against oxidative stress in human colon cells. RESULTS: The obtained data suggest that, among the biocompounds identified in BS, glucosinolates were mainly degraded under gastrointestinal digestion, while phenolics, particularly hydroxycinnamic acid derivatives, were the most resistant constituents. The antioxidant capacity of BS extract subjected to gastrointestinal digestion was similar to or higher than that determined for non-digested BS. Gastrointestinal digested BS extract exhibited reactive oxygen species (ROS)-inhibitory capacity in NCM460 human colon cells, with 1 mg mL(-1) showing an ROS clearance of 76.59%. A 57.33% reduction in oxidative DNA damage in NCM460 cells due to treatment with digested BS extract was observed. CONCLUSION: The results lend support to the possible application of BS as a rich source of antioxidants to improve the defensive system against oxidative stress in the human colon mucosa.

Optimization of the flow cytometry method of detection, quantification and qualification of microorganisms in carrot juice.

Fresh, unpasteurized carrot juice is a popular element of the everyday diet of many consumers, and as such the matter of the juice's microbial safety remains an important one. Imaging flow cytometry (FCM) allows a fast enumeration and determination of cells, as well as their further differentiation. However, carrot juice is a difficult food product to analyze with the use of FCM due to interference from autofluorescence and the presence of plant debris. In this research, we aimed to obtain an effective and repeatable protocol for the preparation of carrot juice samples for FCM analysis. Through experimental and software-based means we successfully determined a reliable protocol for the preparation of fresh, unpasteurized carrot juice, which consisted of a sequence of filtering, centrifugation, enzyme treatment, and finally the implementation of the Machine Learning protocol for the best result.

Global transcriptome analysis of Pseudomonas aeruginosa NT06 response to potassium chloride, sodium lactate, sodium citrate, and microaerophilic conditions in a fish ecosystem.

Pseudomonas aeruginosa is an opportunistic pathogen that recently has been increasingly isolated from foods, especially from minimally processed fish-based products. Those are preserved by the addition of sodium chloride (NaCl) and packaging in a modified atmosphere. However, the current trends of minimizing NaCl content may result in an increased occurrence of P. aeruginosa. NaCl can be replaced with potassium chloride (KCl) or sodium salts of organic acids. Herein, we examined the antimicrobial effects of KCl, sodium lactate (NaL), sodium citrate (NaC), and sodium acetate (NaA) against P. aeruginosa NT06 isolated from fish. Transcriptome response of cells grown in medium imitating a fish product supplemented with KCl and KCl/NaL/NaC and maintained under microaerophilic conditions was analysed. Flow cytometry analysis showed that treatment with KCl and KCl/NaL/NaC resulted in changed metabolic activity of cells. In response to KCl and KCl/NaL/NaC treatment, genes related to cell maintenance, stress response, quorum sensing, virulence, efflux pump, and metabolism were differentially expressed. Collectively, our results provide an improved understanding of the response of P. aeruginosa to NaCl alternative compounds that can be implemented in fish-based products and encourage further exploration of the development of effective methods to protect foods against the P. aeruginosa, underestimate foodborne bacteria.

Simultaneous medium chain carboxylic acids and 1,3-propanediol production in a bioaugmented lactate-based chain elongation induced with glycerol.

The objective was to investigate the impact of the bioaugmentation on chain elongation process using glycerol, lactate and lactose as substrates in an open culture fermentation. In the batch trials the highest selectivity for chain elongation product, i.e. caproate, was observed in trials inoculated with co-culture of Megasphaera elsdenii and Eubacterium limosum grown on glycerol (28.6%), and in non-bioaugmented open culture run on lactose + lactate (14.8%). The results showed that E. limosum, out of two bioaugmented strains, was able to survive in the open culture. A continuous open culture fermentation of glycerol led to caproate and 1,3-propanediol (1,3-PDO) formation, while lactate addition led to 1,3-PDO and short chain carboxylates production. Moving the process into batch mode triggered even-carbon chain elongation. Presence of E. limosum promoted odd-carbon chain elongation and valerate production. Imaging flow cytometry combined with machine learning enabled the discrimination of Eubacterium cells from other microbial strains during the process.

Natural resveratrol analogs differentially target endometriotic cells into apoptosis pathways.

The specific characteristics of endometriotic cells are their ability to evade the apoptotic machinery and abnormal response to apoptotic stimuli. Natural-originated compounds may constitute a beneficial strategy in apoptosis modulation in endometriosis. We investigated and compared the potency of natural resveratrol analogs, including piceatannol, polydatin, and pterostilbene, in targeting cell death pathways, including apoptosis-related morphologic and biochemical processes, alongside the modulation of the critical genes expression. Upon resveratrol and pterostilbene treatment, a significant reduction of endometriotic cell viability and an increased apoptotic proportion of cells were noted. The lower antiproliferative potential was found for piceatannol and polydatin. Endometrial stromal T HESC cells were significantly more resistant than endometriotic epithelial 12Z cells to the cytotoxic activity of all analyzed compounds. They differentially affected endometriotic cell viability, cell cycle, anti- and proapoptotic genes regulation, caspases expression and enzymatic activity, and DNA fragmentation. Pterostilbene-mediated endometriotic cell apoptosis modulation was confirmed to be most effective but without evident caspase 3 upregulation. Our study provides valuable insight into the apoptogenic activity of resveratrol and its natural analogs in endometriotic cells. Data obtained revealed the highest therapeutic potential of pterostilbene by effectively targeting cell death determinants in endometriosis, strengthening its optimization in further extensive research.

Anti-proliferative potential and oxidative reactivity of thermo-oxidative degradation products of stigmasterol and stigmasteryl esters for human intestinal cells.

Stigmasterol in free and esterified form is incorporated in LDL cholesterol-lowering food products, intended for direct consumption and cooking, baking, and frying. Under thermal treatment, stigmasterol compounds may constitute a source of thermo-oxidative degradation products and oxyderivatives with potentially adverse health effects. This study aimed to analyze the anti-proliferative potential and genotoxicity of thermo-oxidatively treated stigmasterol (ST), stigmasteryl linoleate (ST-LA), and oleate (ST-OA). The effects on cell viability and proliferation, cell cycle progression, intracellular reactive oxygen species (ROS) generation, and DNA damage were analyzed in normal human intestinal cells. The mutagenic potential was assessed in a bacterial reverse mutation test using Salmonella enterica serovar Typhimurium strains involving metabolic activation. Stigmasteryl esters showed a significantly lower potential to affect intestinal cell viability and proliferation than non-esterified ST, regardless of heating. Thermo-oxidatively treated ST suppressed intestinal cell proliferation by arresting the cell cycle in the G2/M phase and DNA synthesis inhibition. The enhanced intracellular ROS generation and caspase 3/7 activity suggest targeting intestinal cells to the apoptosis pathway. Also, heated ST-LA intensified ROS production and elicited pro-apoptotic effects. Thermo-oxidative derivatives of ST and ST-LA may evoke harmful gastrointestinal effects due to their high oxidative reactivity towards intestinal cells.

Chlorogenic Acid Inhibits Rahnella aquatilis KM25 Growth and Proteolytic Activity in Fish-Based Products.

This work verified the antiproliferative and antiproteolytic activities of chlorogenic acid against Rahnella aquatilis KM25, a spoilage organism of raw salmon stored at 4 °C. Chlorogenic acid limited the growth of R. aqatilis KM25 in vitro at a concentration of 2.0 mg/mL. The dead (46%), viable (25%), and injured (20%) cell subpopulations were identified by flow cytometry following treatment of R. aquatilis KM25 with the examined agent. The exposure of R. aquatilis KM25 to chlorogenic acid altered its morphology. Changes in cell dimensions, mostly in length parameters from 0.778 µm to 1.09 µm, were found. The length of untreated cells ranged from 0.958 µm to 1.53 µm. The RT-qPCR experiments revealed changes in the expression of genes responsible for the proliferation and proteolytic activity of cells. Chlorogenic acid caused a significant reduction in the mRNA levels of the ftsZ, ftsA, ftsN, tolB, and M4 genes (-2.5, -1.5, -2.0, -1.5, and -1.5, respectively). In situ experiments confirmed the potential of chlorogenic acid to limit bacterial growth. A similar effect was noted in samples treated with benzoic acid, where the growth inhibition of R. aquatilis KM25 was 85-95%. Reduction of microbial R. aquatilis KM25 proliferation significantly limited total volatile base nitrogen (TVB-N) and trimethylamine (TMA-N) formation during storage, extending the shelf life of model products. The TVB-N and TMA-N parameters did not exceed the upper levels of the maximum permissible limit of acceptability. In this work, the TVB-N and TMA-N parameters were 10-25 mg/100 g and 2.5-20.5 mg/100 g, respectively; for samples with benzoic acid-supplemented marinades, the parameters TVB-N and TMA-N were 7.5-25.0 mg/100 g and 2.0-20.0 mg/100 g, respectively. Based on the results of this work, it can be concluded that chlorogenic acid can increase the safety, shelf life, and quality of fishery products.

Multi-faceted analysis of bacterial transformation of nitrofurantoin.

Excessive presence of antibiotics and their residues can be dangerous to the natural environment. To reduce this negative effect, efficient strategies to remove them from the ecosystem are required. This study aimed to explore the potential of bacterial strains to degrade nitrofurantoin (NFT). Single strains isolated from contaminated areas, namely Stenotrophomonas acidaminiphila N0B, Pseudomonas indoloxydans WB, and Serratia marcescens ODW152 were employed in this study. Degradation efficiency and dynamic changes within the cells during NFT biodegradation were investigated. For this purpose, atomic force microscopy, flow cytometry, zeta potential, and particle size distribution measurements were applied. Serratia marcescens ODW152 showed the highest performance in removal of NFT (96 % in 28 days). The AFM images revealed modifications of cell shape and surface structure induced by NFT. Zeta potential showed significant variations during biodegradation. Cultures exposed to NFT had a broader size distribution than the control cultures due to increased cells agglomeration or aggregation. 1-Aminohydantoin and semicarbazide were detected as nitrofurantoin biotransformation products. They showed increased cytotoxicity toward bacteria as determined by spectroscopy and flow cytometry. Results of this study suggest that nitrofurantoin biodegradation leads to formation of stable transformation products that significantly affect the physiology and structure of bacterial cells.

The Effects of Cellular Membrane Damage on the Long-Term Storage and Adhesion of Probiotic Bacteria in Caco-2 Cell Line.

Adhesion is one of the main factors responsible for the probiotic properties of bacteria in the human gut. Membrane proteins affected by cellular damage are one of the key aspects determining adhesion. Fluid-bed-dried preparations containing probiotic bacteria were analyzed in terms of their stability (temperature of glass transition) and shelf life in different conditions (modified atmosphere, refrigeration). Imaging flow cytometry was utilized to determine four subpopulations of cells based on their physiological and morphological properties. Lastly, adhesion was measured in bacteria cultured in optimal conditions and treated with heat shock. The results show that the subpopulations with no or low levels of cell membrane damage exhibit the ability to adhere to Caco-2 cells. The temperature of protein denaturation in bacteria was recorded as being between 65 °C and 70 °C. The highest glass transition temperature (Tg) value for hydroxypropyl methylcellulose (used as a coating substance) was measured at 152.6 °C. Drying and coating can be utilized as a sufficient treatment, allowing a long shelf-life (up to 12 months). It is, however, worth noting that technological processing, especially with high temperatures, may decrease the probiotic value of the preparation by damaging the bacterial cells.

Dynamics of Active Fluorescent Units (AFU) and Water Activity (aw) Changes in Probiotic Products-Pilot Study.

The flow cytometry method (FCM) is a widely renowned practice increasingly used to assess the microbial viability of probiotic products. Additionally, the measurement of water activity (aw) can be used to confirm the presence of viable cells in probiotic products throughout their shelf lives. The aim of this study was to investigate the correlation between changes in aw and variations in active fluorescent units (AFU), a unit commonly used in flow cytometry method, during the aging of probiotic products containing freeze-dried bacteria. We controlled the stability of probiotic products for bacterial counts (using ISO 19344 method) and aw levels in commercially available capsules containing freeze-dried bacteria such as Lactobacillus sp. or combinations of Lactobacillus sp. and Bifidobacterium sp. in standard conditions (25 ± 2 °C and 60% relative humidity) over a period of 24 months. During this time, the bacterial contents decreased by 0.12 Log10 in the single-strain product, by 0.16 Log10 in the two-strain product and by 0.26 Log10 in the multi-strain product. With the increase in aw, the number of bacteria decreased but the aw at the end point of the stability study did not exceed 0.15 in each of the three tested products. FCM combined with aw is a prospective analysis that can be used to assess the stability of probiotic products, both for its ability to detect bacterial viability and for practical (analysis time) and economic reasons.

In silico and in vitro analysis of the impact of single substitutions within EXO-motifs on Hsa-MiR-1246 intercellular transfer in breast cancer cell.

MiR-1246 has recently gained much attention and many studies have shown its oncogenic role in colorectal, breast, lung, and ovarian cancers. However, miR-1246 processing, stability, and mechanisms directing miR-1246 into neighbor cells remain still unclear. In this study, we aimed to determine the role of single-nucleotide substitutions within short exosome sorting motifs - so-called EXO-motifs: GGAG and GCAG present in miR-1246 sequence on its intracellular stability and extracellular transfer. We applied in silico methods such as 2D and 3D structure analysis and modeling of protein interactions. We also performed in vitro validation through the transfection of fluorescently labeled miRNA to MDA-MB-231 cells, which we analyzed by flow cytometry and fluorescent microscopy. Our results suggest that nucleotides alterations that disturbed miR-1246 EXO-motifs were able to modulate miRNA-1246 stability and its transfer level to the neighboring cells, suggesting that the molecular mechanism of RNA stability and intercellular transfer can be closely related.