Search

Background: Inflammation and immune cell dysfunction are critical facilitators of endometriosis pathophysiology. Macrophages are renowned for stimulating lesion growth, vascularization, innervation, and pain generation. By combining macrophages and endometriotic cells, we determined if resveratrol and its natural analogs can target the immune dysregulation and oxidative imbalance in endometriosis. Methods: After treatment with compounds (5, 10, 25 µM), we evaluated the expression of key inflammatory and oxidative stress markers, cytokines release, and ROS production by applying q-PCR, ELISA, Cytometric Beads Array, and multiplexed fluorogenic staining and flow cytometry analysis with bioimaging. Results: The results showed that endometriosis-related macrophages treated with stilbenes have impaired expression of pro-inflammatory markers ( IL6 , IL8 , IL1B , TNF , CCL2 , CXCL10 , PTGS2 ). The effect of resveratrol, pterostilbene, and piceatannol was observed, especially in reducing IL1B , CCL2 , and CXCL10 genes up to 3.5-, 5-, and 7.7-fold at 25 µM, respectively. Also, with piceatannol or polydatin exposure, the IL-6 decrease was noticeable. This study reported an antioxidant effect by reducing ROS-positive cells from 96% to 48% by pterostilbene. Results from flow cytometry correlated with the transcript activation of detoxification enzymes ( SOD , GPX ). Conclusions: Prospects for potential therapy based on regulating the immune microenvironment and reducing the accumulation of free radicals with stilbenes application were described in the article.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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 (a w ) 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 a w 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 a w 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 Log 10 in the single-strain product, by 0.16 Log 10 in the two-strain product and by 0.26 Log 10 in the multi-strain product. With the increase in a w , the number of bacteria decreased but the a w at the end point of the stability study did not exceed 0.15 in each of the three tested products. FCM combined with a w 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.

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.

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., (© 2023. The Author(s).)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 G 2 /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., (© 2023. The Author(s).)

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.

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., (© 2022. The Author(s).)

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.

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., (© 2021. The Author(s).)

Atmospheric cold plasma (ACP) inactivation of Lentilactobacillus hilgardii was investigated. Bacteria were exposed to ACP dielectric barrier discharge with helium and oxygen as working gases for 5, 10, and 15 min. The innovative approach in our work for evaluation of bacterial survival was the use in addition to the classical plate culture method also flow cytometry which allowed the cells to be sorted and revealed different physiological states after the plasma treatment. Results showed total inhibition of bacterial growth after 10-min of ACP exposure. However, the analysis of flow cytometry demonstrated the presence of 14.4% of active cells 77.5% of cells in the mid-active state and 8.1% of dead cells after 10 min. In addition, some of the cells in the mid-active state showed the ability to grow again on culture medium, thus confirming the hypothesis of induction of VBNC state in L .hilgardii cells by cold plasma. In turn, atomic force microscopy (AFM) which was used to study morphological changes in L. hilgardii after plasma treatment at particular physiological states (active, mid-active, dead), showed that the surface roughness of the mid-active cell (2.70 ± 0.75 nm) was similar to that of the control sample (2.04 ± 0.55 nm). The lack of considerable changes on the cell surface additionally explains the effective cell resuscitation. To the best of our knowledge, AFM was used for the first time in this work to analyze cells which have been sorted into subpopulations after cold plasma treatment and this is the first work indicating the induction of VBNC state in L. hilgardii cells after exposure to cold plasma.

The objective of this study was to investigate the effect of substrate composition on chain elongation pathways and on shaping reactor microbiome during open culture fermentation (OCF). The process was performed in a continuous mode in an upflow anaerobic sludge blanket (UASB) reactor fed with either fresh acid whey (AW) or AW at controlled stage of prefermentation (with controlled content of electron donors). Dosing AW with an increasing ethanol loading rate led to ethanol oxidation and short chain carboxylic acids (SCCAs) generation. Change of the feedstock composition (higher lactate and lactose content and ethanol cut off) shifted the process outcome towards medium chain carboxylic acids (MCCAs) production, with caproate as the main product. The MCCAs production rate has grown from 0.7 ± 0 to 4.12 ± 1 g/L/day (38.3 ± 5 to 212.6 ± 60 mmol C/L/day) and reached specificity of 48 ± 18% mol C. The differentiation between microbiome samples confirmed the reactor microbiome shaped according to the feed composition. The only known caproic acid producers were represented by Caproiciproducens ssp., that reached a relative OTU abundance between 3 and 7%. The developed method enables to substitute the use of fossil resources with products from the OCF of waste and wastewater. Thus, it contributes to reduce the carbon footprint and enhance the sustainability of the chemical industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Thirty nine strains of Galactomyces geotrichum molds were isolated from a traditional fried cottage cheese and production of polyunsaturated fatty acids (PUFA) was assessed. Among them eleven strains produced an extracellular lipids enriched in n-6 and n-3 PUFA. The extracellular lipids produced by G. geotrichum strain 38 contained the highest amounts of total PUFA (24.3%), with the highest contribution of n-3 fatty acids (17.9%), where α-linolenic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids were the main contributors. To obtain maximal production of PUFA, composition of the medium consisted of 10 g/L rapeseed oil, 5 g/L yeast extract, 0.05 g/L K 2 HPO 4 , 0.17 g/L MgSO 4 , 0.015 g/L MnSO 4 , 0.015 g/L ZnSO 4 , 0.05 g/L FeSO 4 , and 10 mg/L vitamin B 12 . The optimal growth conditions at 30 °C involve: aeration at 1.5 vvm (volume of air per volume of broth per minute) at pH 6.5. The cheese produced under described conditions contained higher amount of n-3 PUFA (0.25 mg/g cheese) in comparison to control (0.01 mg/g). α-Linolenic acid predominated among n-3 fatty acids. Galactomyces geotrichum is a natural microflora of dairy products, and could be used to enrich food/cheese in deficient omega-3 lipids., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Essential oils can be used as preservatives in foods because of their ability to inhibit bacteria growth in low concentration, which does not influence on foods' organoleptic properties and does not generate the resistance mechanisms in cells. The aim of that work was to compare the effectiveness of commercial oils from black pepper ( Piper nigrum ), rosemary ( Rosmarinus officinalis ), lemongrass ( Cymbopogon citratus ) and juniper ( Juniperus communis L.) with oils obtained in our laboratory. The typical cultivation method was supported by the flow cytometry to detect the cells of very low physiologic and metabolic activity. Our investigation demonstrated that both types of oils can effectively inhibit the growth of saprophytic bacteria P. orientalis . The oils distilled in our laboratory had a bacteriostatic effect at a lower concentration, which is important for application in the food industry. Flow cytometry analyzes and confirmed the thesis that essential oils do not have a germicidal effect on bacteria cells., Competing Interests: The authors declare no conflicts of interest.

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., Competing Interests: Polymerase Chain Reaction

The aim of this work was to estimate the antibacterial activity of selected essential oils on Pseudomonas orientalis strains isolated from foods. An attempt was also made to identify the mechanisms of the action of the plant oils. Classical methods of assessment of the effectiveness of antimicrobial activity of oils were linked with flow cytometry. It was observed that bergamot, lemongrass, bitter orange, juniper, and black pepper oils have bacteriostatic effect against P. orientalis P49. P. orientalis P110 is sensitive to lime, lemongrass, juniper, rosemary, and black pepper oils. Additionally, plant oils with biostatic effect on P. orientalis limited the intracellular metabolic activity of cells; this was closely linked with the ability of plant oils' bioactive components to interact with bacteria cell membrane, causing the release of membrane proteins. As a result, the selective permeability of the cell membranes were damaged and the bacterial shape was transformed to coccoid in form.

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.

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.

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in the first exon of the huntingtin gene ( HTT ). The accumulation of polyglutamine-rich huntingtin proteins affects various cellular functions and causes selective degeneration of neurons in the striatum. Therapeutic strategies used to date to silence the expression of mutant HTT include antisense oligonucleotides, RNA interference-based approaches and, recently, genome editing with the CRISPR/Cas9 system. Here, we demonstrate that the CAG repeat tract can be precisely excised from the HTT gene with the use of the paired Cas9 nickase strategy. As a model, we used HD patient-derived fibroblasts with varied numbers of CAG repeats. The repeat excision inactivated the HTT gene and abrogated huntingtin synthesis in a CAG repeat length-independent manner. Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for HD that can be extended to other polyQ disorders.

An intensive investigation of the development of in vitro models to study tumor biology has led to the generation of various three-dimensional (3D) culture methods that better mimic in vivo conditions. The tumor microenvironment (TME) is shaped by direct interactions among cancer cells, cancer-associated cells and the extracellular matrix (ECM). Recognizing the need to incorporate both tissue dimensionality and the heterogeneity of cells, we have developed a 3D breast cancer model. NIH3T3 fibroblasts and EMT6 breast cancer cell lines were seeded in various ratios onto a silk fibroin scaffold. The porosity of the silk scaffold was optimized to facilitate the growth of cancer cells. EMT6 and NIH3T3 cells were modified to express GFP and turboFP635, respectively, which enabled the direct analysis of the cell morphology and colonization of the scaffold and for the separation of the cells after their co-culture. Use of 3D mono-culture and 3D co-culture methods resulted in the modification of cell morphology and in a significant increase in ECM production. These culture methods also induced cellular changes related to EMT (epithelial-mesenchymal transition) and CAF (cancer-associated fibroblast) markers. The presented model is an easy to manufacture, well-characterized tool that can be used to study processes of the TME., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.