DNA Dye Research Articles

A convenient approach for generating dimeric nucleic acid dyes via click-chemistry

Fluorescent dyes are essential tools for visualizing DNA and RNA. Dimeric dyes like GelGreen have gained popularity as safer alternatives to ethidium bromide (EB) due to their reduced mutagenicity and genotoxicity. In this study, we present a straightforward method to synthesize novel acridine orange (AO)-based dimeric dyes using click chemistry. Starting from acridine orange, these dyes can be synthesized in just two steps. Compared to GelGreen, these new dyes incorporate additional triazole groups in their linkers. They exhibit a maximum absorption wavelength of approximately 472 nm, which shifts to around 503 nm upon binding with DNA, allowing excitation by blue light. These dyes show minimal fluorescence in aqueous solutions, indicating that they adopt a closed conformation where the fluorescence of acridine orange is quenched due to intramolecular aggregation. The presence of DNA significantly enhances their fluorescence at around 526 nm, suggesting that DNA binding induces an open conformation. This “light-up” property makes them highly sensitive DNA dyes with a strong signal-to-noise ratio. We successfully applied these novel dyes in agarose gel electrophoresis, where they demonstrated excellent performance.

Insights into the genomic and phenotypic characteristics of Bacillus spp. strains isolated from biofilms in broiler farms.

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.

Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils.

With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSChighFSChighCD15+CD66b+. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR+7-AAD+) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIRdim7-AAD+) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins - myeloperoxidase (MPO) and neutrophil elastase (NE) - were utilized to identify neutrophil-appendant NET events (SSChighFSChighCD15+CD66b+Zombie NIRdim7-AAD+MPO+NE+). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.

Design and synthesis of large Stokes shift DNA dyes with reduced genotoxicity

Despite intensive search over the past decades, only a few small-molecule DNA fluorescent dyes were found with large Stokes shifts. These molecules, however, are often too toxic for widespread usage. Here, we designed DNA-specific fluorescent dyes rooted in benzimidazole architectures with a hitherto unexplored molecular framework based on thiazole-benzimidazole scaffolding. We further incorporated a pyrazole ring with an extended sidechain to prevent cell penetration. These novel benzimidazole derivatives were predicted by quantum calculations and subsequently validated to have large Stokes shifts ranging from 135 to 143 nm, with their emission colors changed from capri blue for the Hoechst reference compound to iguana green. These readily-synthesized compounds, which displayed improved DNA staining intensity and detection limits along with a complete loss of capability for cellular membrane permeation and negligible mutagenic effects as designed, offer a safer alternative to the existing high-performance small-molecule DNA fluorescent dyes.

A proposal for new genotoxic and cytotoxic endpoints to assess chemical effects on the red algae Ceramium tenuicorne.

Macroalgae provide key contributions to aquatic ecosystems, including their role as primary producers and the provision of biotopes for marine organisms, fish spawning, and fish nurseries. The aim of this study was to evaluate the feasibility of a micronucleus test and a comet assay in Ceramium tenuicorne, a red macroalga. This alga is widely distributed in marine ecosystems and brackish waters, and is therefore a potential bioindicator of the effects of anthropogenic pollution in these ecosystems. Unfortunately, the two genotoxicity tests evaluated were not suitable for this alga because the nuclei are generally very small (between 2 and 10µm), are variable in size, and there may be several nuclei in each cell (between 1 and 5 in our observations). However, the present study allowed us to define conditions for observing these algal cells and optimizing the choice of DNA dye (orcein), cell fixation solution (Carnoy's solution), and hydrolysis step (HCl, 1200mmol/L solution for 1min). This research allowed us to propose two genotoxicity and cytotoxicity endpoints for assessing chemical effects on the algal cells: counting of nuclei in cortical cell areas, and the frequency of axial cells in mitosis. These two criteria were measured after exposing C. tenuicorne to two reference substances: cadmium chloride and maleic hydrazide, and we highlight the effects from 1 × 10-5M of CdCl2 and 5 × 10-5M of maleic hydrazide.

Prediction of cell cycle distribution after drug exposure by high content imaging analysis using low-toxic DNA staining dye.

Interference in cell cycle progression has been noted as one of the important properties of anticancer drugs. In this study, we developed the cell cycle prediction model using high-content imaging data of recipient cells after drug exposure and DNA-staining with a low-toxic DNA dye, SiR-DNA. For this purpose, we exploited HeLa and MCF7 cells introduced with a fluorescent ubiquitination-based cell cycle indicator (Fucci). Fucci-expressing cancer cells were subjected to high-content imaging analysis using OperettaCLS after 36-h exposure to anticancer drugs; the nuclei were segmented, and the morphological and intensity properties of each nucleus characterized by SiR-DNA staining were calculated using imaging analysis software, Harmony. For the use of training, we classified cells into each phase of the cell cycle using the Fucci system. Training data (n = 7500) and validation data (n = 2500) were randomly sampled and the binary classification prediction models for G1, early S, and S/G2/M phases of the cell cycle were developed using four supervised machine learning algorithms. We selected random forest as the model with the best performance through 10-fold cross-validation; the accuracy rate was approximately 75%-87%. Regarding feature importance, variables expected to be biologically related to the cell cycle, for example, signal intensity and nuclear size, were highly ranked, suggesting the validity of the model. These results showed that the cell cycle can be predicted in cancer cells by simply exploiting the current prediction model using fluorescent images of DNA-staining dye, and the model could be applied for the use of future exvivo drug sensitivity diagnosis.

Abstract 1631: A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture

Abstract We describe a novel homogeneous cell-based assay developed for the quantitative detection of Ki-67 protein, a recognized marker of cell proliferation, utilizing Lumit™ immunoassay technology. This luminescent assay has been effectively multiplexed with a fluorescent measure of cell death, employing a cell-impermeant fluorogenic DNA dye. This multiplex allows for the clear distinction between antiproliferative activity and overt cytotoxicity in cell culture models. Through varying lytic conditions, we demonstrated effective cell lysis and detection of nuclear Ki-67 protein. Specifically, Lumit™ Lysis Buffer II provided efficient recovery of Ki-67 protein without compromising assay performance, including the requisite binding of labeled antibodies to the target protein. This assay exhibited excellent signal-to-background (up to 40-fold) and signal linearity (R^2 = 0.99) in wildtype HeLa cells (~1000 to 80,000/well), while the signal in Ki-67 knockout HeLa cells was comparable to the no-cell background, indicating excellent specificity for Ki-67 protein. Due to the unavailability of recombinant full-length Ki-67 protein, two different recombinant Ki-67 protein fragments were used to corroborate assay sensitivity of <1 pM Ki-67 protein. The assay's suitability for human Ki-67 protein detection, not mouse, was observed through further cell-based testing. The proliferation/cytotoxicity assay multiplex was validated in both suspension and adherent cell lines, as well as primary cell culture, detecting antiproliferative and proliferative responses. CDK4/6 inhibitors are a class of antiproliferative agents of significant interest for their potential in treating cancer. In Jurkat cells, treatment with the CDK4/6 inhibitor palbociclib led to a dose-dependent reduction in Ki-67 protein expression with an EC50 of 88 nM, and total Ki-67 elimination at 2 μM compound concentration. Notably, there was no cytotoxicity detected at the tested doses and treatment duration (up to 48 h), but a significant reduction in viable cell number at maximal palbociclib concentrations, aligning with an antiproliferative mechanism. This work establishes a rapid, simple, luminescent assay for determining Ki-67 levels that can be paired with a fluorogenic readout of cell death, facilitating efficient and quantitative discernment of altered proliferation in mammalian cell culture using standard microplate readers. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1631.

Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining.

In the adult mouse testis, germ cells of various developmental cell states co-exist. FACS isolation of cells stained with the DNA dye Hoechst 33342 has been used for many years to sub-divide these cells based on their DNA content. This approach provides an efficient way to obtain broad categories of male germ cells: pre-meiotic spermatogonia, meiotic spermatocytes and post-meiotic spermatids. The addition of a red filter for Hoechst staining enables further sub-division of spermatocytes depending on sub-stages of meiotic prophase. However, separation of different stage spermatids using Hoechst staining alone is not possible. We recently reported a methodology, combining Hoechst staining with a second DNA dye (SYTO16) that enables the further separation of these cells into three sub-populations: round, early elongating, and late elongating spermatids (Gill et al., Cytometry A 101:529-536, 2022). This method makes it possible to obtain rapidly and simply pure fractions of male germ cells from multiple developental stages from the same animal.

Cyrene™ as a green alternative to N,N'-dimethylformamide (DMF) in the synthesis of MLCT-emissive ruthenium(II) polypyridyl complexes for biological applications.

Ruthenium(II) polypyridyl complexes (RPCs) that emit from triplet metal-to-ligand charge transfer (MLCT) states find a wide variety of uses ranging from luminophores to potential anti-cancer or anti-bacterial therapeutics. Herein we describe a greener, microwave-assisted synthetic pathway for the preparation of homoleptic [Ru(N^N)3]2+ and bis-heteroleptic [Ru(N^N)2(N'^N')]2+ type complexes. This employs the bio-renewable solvent Cyrene™, dihydrolevoglucosenone, as a green alternative to N,N'-dimethylformamide (DMF) in the synthesis of Ru(N^N)2Cl2 intermediate complexes, obtaining comparable yields for N^N = 2,2'-bipyridine, 1,10-phenanthroline and methylated derivatives. Employing these intermediates, a range of RPCs were prepared and we verify that the ubiquitous luminophore [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) can be prepared by this two-step green pathway where it is virtually indistinguishable from a commercial reference. Furthermore, the novel complexes [Ru(bpy)2(10,11-dmdppz)]2+ (10,11-dmdppz = 10,11-dimethyl-dipyridophenazine) and [Ru(5,5'-dmbpy)2(10,11-dmdppz)]2+ (5,5'-dmbpy = 5,5'-dimethyl-bpy) intercalate duplex DNA with high affinity (DNA binding constants, Kb = 5.7 × 107 and 1.0 × 107 M-1, respectively) and function as plasma membrane and nuclear DNA dyes for confocal and STED microscopies courtesy of their long-lived MLCT luminescence.

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

(Very) Small Stem-like Cells in Human Cell Cultures

Very Small Embryonic-like Stem Cells (VSELSCs) and Very Small Cancer Stem Cells (VSCSCs) are fields of intensive research. Although the presence in vitro of VSELSC and VSCSC cellular stage analogs appear probable, it has yet to be published. Utilizing established human cell cultures with varying populations of primitive cells, stained with CD markers specific to primitive stages, in addition to a fluorescent DNA dye, and following histochemical processing, we have developed a cytological method for detecting Very Small Leukemic Stem-like Cells (VSLSLCs), Very Small Cancer Stem-like Cells (VSCSLCs), and VSELSCs. This detection provides an opportunity to advance research in these areas.

Effect of ionic strength on DNA–dye interactions of Victoria blue B and methylene green using UV–visible spectroscopy

Abstract Victoria blue and methylene green dyes have both been extensively studied due to their numerous applications, including their ability to bind to DNA. Dyes are very important in everyday life with applications in textile, cosmetics, food and pharmaceutical industries. It has been found that some of them adversely affect human health causing severe abnormalities. Among these abnormalities, cancer is of great concern due to its fatal and almost non-recoverable nature. In this work we have studied the binding of two dyes namely Victoria blue B (VBB) and Methylene green (MG) with double stranded DNA (Salmon sperm). The interactions were studied in the presence of different concentrations of buffer solutions at a constant pH. The selected dyes showed interactions with double-stranded DNA through intercalation and electrostatic modes. Upon increasing ionic strength of the buffer the binding constant (K b ) value for MG was decreased whereas increased for VBB, which conclude that, at higher ionic strength (0.5 M) the DNA–MG interactions is lower and DNA–VVB interactions is maximum. The carcinogenicity of a given dye is indicated from its binding constants in the current study. Based on the recorded K b values of the selected dyes it was concluded that proper disposing and precautions should be taken while utilizing/dealing these dyes in order to minimize/avoid their impact on environment and human health.

Cell Counting Techniques for Preimplantation Mouse Embryos Using Fluorescent DNA Dyes.

Counting cells in preimplantation embryos by light microscopy is straightforward until morula compaction, when cell boundaries in living embryos become indistinct. An alternative to morphological assessment of cell number is to use fluorescent DNA dyes. This protocol details simple nuclear counting with a single DNA dye (Hoechst) or a more complicated procedure in which differential nuclear counts of the trophectoderm and inner cell mass (ICM) can be made using immunosurgery of the blastocyst and two DNA dyes (Hoechst and propidium iodide).

Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase

AbstractActivated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell’s DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Trolox Antioxidant as a Factor in Stabilization of Human Cord Blood Nucleated Cells During Cryopreservation

The paper presents experimental data on the determination of preservation rate of human cord blood nucleated cells and their apoptosis/necrosis stages to determine the number of viable functionally active cells after cryopreservation in solutions with different concentrations of DMSO and the water-soluble analogue of vitamin E, the antioxidant trolox. Counting the number of human cord blood nucleated cells after freezing in media with the addition of trolox revealed their maximum preservation in the samples with 7.5% DMSO and 50, 70 or 200 μM of the antioxidant. Using the flow cytometry with the addition of the Annexin V FITC reagent, which specifically binds to phospholipids, and the DNA dye 7-amino-actinomycin D (7-AAD), it was established that trolox in concentrations of 50–70 μM provided an increase in the number of viable cells with intact membrane (AnnexinV─/7AAD─) by 12–16% compared to the control, which involved the use of only DMSO in the cryoprotective solution. The obtained results indicate the effectiveness of using the antioxidant trolox and the prospects of developing trolox-containing cryoprotective mixtures for freezing and long-term storage of nucleated cord blood cells, including hematopoietic progenitor cells.

The effect of sex-sorting on stallion semen quality

Spermatozoa retain fertilizing potential after sorting, but separation into X and Y populations has deleterious effects on the cell (Samper JC et al. Clinical Theriogenology 2022). The effect of sex-sorting (SS) on stallion sperm was evaluated in single ejaculates from 32 fertile, sexually active stallions. Based on previous work (Samper et al 2022) Botu-Gold was selected to dilute 2 billion cells at 1:3-1:4 ratio. Extender was supplemented with the DNA dye Hoechst 33342 and cooled at 8 ̊C for 18-24 h. Semen was warmed to 20 ̊C, and centrifuged through EquiPure at 200g for 30 min. The pellet was resuspended at 250-300 million/mL using ST Equiship, ST Genetics Texas) and transported at ∼20°C to the sorting facility. Sperm were SS (SexedULTRA™ Genesis III flow cytometer). Purity gate was set at 95% for both genders. Percent morphologically normal (%NORM) (Nomarski 1000X); percent total motility (TMOT), curvilinear velocity (VCL [um/sec]) (IVOS II); percent viability (VIAB; NucleoCounter) were evaluated at times, T1-after cooling, T2- after Equipure, T3- 0 to 6hs after sorting. Semen was cooled to 8°C and TMOT, VCL and VIAB were assessed at 18-24h (T4) and 48h (T5) post-sorting. Treatment means at each treatment/time were analyzed by a repeated measures mixed-model ANOVA. Sort quality measures (% DEAD) Peak Valley Ratio [PVR] and % percent of sperm correctly oriented [ORT]) were correlatedwith %NORM. Although there was a wide variation between stallions, 29/32 (91%) stallions were successfully sorted. The %NORM (mean±sd) was 59±11, 74±9 and 77±9% for T1, T2 and T3 respectively (p<0.05). For TMOT, T1 (67%) was less than T2 (74%; P<0.05) and similar to T3 (71%; P>0.05); for VIA, T1 (74%) was less than T2 and T3 (79 and 80%; P<0.05); for VCL, T1 (157µ/s) was less than T2 (171 µ/s; P<0.05) and similar to T3 (166 µ/s; P>0.05). For sperm longevity (TMOT, VIA, and VCL) T4 (49, 73, 147) and T5 (27, 63, 117) decreased, compared to T3. The initial quality of the semen and the number of morphological abnormalities had a high impact on the sorting and quality parameters ORT and PVR were positively correlated while DEAD was negatively correlated with %Norm cells in T1, T2 and T3. In this study, the SS process (cooling, colloid centrifugation and SS) occurred over an ∼ 24-hour period and the quality of the SS sperm was improved and maintained over that period which is similar to those normally utilized for conventional cool-shipped semen programs. Current sex-sorting technology has improved significantly, resulting in a high number of stallions sorting successfully, allowing broad application of gender selection technology to the equine breeding industry.

On binding mechanism exploration of organic amphoteric dye Rhodamine B with natural polymeric DNA

Nucleic acids, like DNA and RNA, are molecules that are present in any life form. Their most notable function is to encode biological information. DNA also has unique material properties, which attract material scientists, nanotechnologists, and engineers. The study and investigation of the structural and dynamical properties of complex biological systems have attracted considerable interest among scientists in general and physicists and biologists in particular.The alteration in DNA due to the introduction of any small molecule changes the overall function and stability of DNA. So quantitative information about the effects of such ligand binding on the structure and function of DNAs, are very much required. In this study, the interaction between natural polymeric DNA (deoxyribonucleic acid sodium salt from herring testes; derived from the sperm of herring fish) and organic amphoteric dye molecule (fluorescent, biomarker), Rhodamine B, has been studied quantitatively. An overview of the overall changes occurring in the macromolecule was estimated using multi-spectroscopic methods and complemented with computational analyses. The results obtained provide an understanding of the binding affinity, stoichiometry, and structural details of the ligand-DNA complex, followed by an estimation of associated binding thermodynamics. Finally, structure–function relationships and molecular aspects of ligand binding to DNA have been established in the current piece of research work, which can have multifaceted applications in drug development and pharmacology.

Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye

Bacteriophages (phages) are viruses that specifically attack bacteria. Their use as therapeutics, which constitutes a promising alternative to antibiotics, heavily relies on selecting effective lytic phages against the pathogen of interest. Current selection techniques are laborious and do not allow for direct visualization of phage infection dynamics. Here, we present a method that circumvents these limitations. It can be scaled for high-throughput and permits monitoring of the phage infection in real time via a fluorescence signal readout. This is achieved through the use of a membrane-impermeant nucleic acid dye that stains the DNA of damaged or lysed bacteria and new phage progeny. We have tested the method on Pseudomonas aeruginosa and Klebsiella pneumoniae and show that an increase in fluorescence reflects phage-mediated killing. This is confirmed by other techniques including spot tests, colony plating, flow cytometry and metabolic activity measurements. Furthermore, we illustrate how our method may be used to compare the activity of different phages and to screen the susceptibility of clinical isolates to phage. Altogether, we present a fast, reliable way of selecting phages against Gram-negative bacteria, which may be valuable in optimizing the process of selecting phages for therapeutic use.

Label-free virtual staining of neutrophil extracellular traps (NETs) in microfluidics.

Neutrophils are the most abundant circulating white blood cells and one of their critical functions to eliminate pathogenic threats includes the release of extracellular DNA, also known as neutrophil extracellular traps (NETs), which is dysregulated in many diseases including cancer, type 2 diabetes mellitus and infectious diseases. Currently, conventional methods to quantify the NET formation (NETosis) rely on fluorescence antibody-based NET labelling or circulating NET-associated protein detection by ELISA, which are expensive, laborious, and time-consuming. In this work, we employed a novel "virtual staining" using deep convolutional neural networks (CNNs) to facilitate label-free quantification of NETs trapped in a micropillar array in a microfluidic device. Virtual staining is constructed to establish relations between morphological features in phase contrast images and fluorescence features in Sytox-green (DNA dye) images. We first investigated the effect of different learning rates on model training and optimized the learning rate to achieve the best model which can provide outputs close to Sytox green staining based on various reconstruction metrics (e.g., structural similarity (SSIM) and pixel-wise error (MAE, MSE)). The virtual staining of different NET concentrations was investigated which showed a linear correlation with fluorescent staining. As a proof of concept for clinical testing, the model was used to characterize purified neutrophils treated with NETosis inducers, including lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore (CaI), and successfully detected different NET profiles for different treatments. Collectively, these results demonstrated the potential of using deep learning for enhanced label-free image analysis of NETs for clinical research, drug discovery and point-of-care testing of diseases.