Nucleic Acid Stain Research Articles

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF–promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF–promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF–promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF–promoter binding assay, which not only complements currently available approaches for DNA–protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria–host interface.

DIPG-29. Highly multiplexed digital spatial profiling of the tumor immune environment of pediatric and adultH3 K27M-mutant diffuse midline gliomas

Abstract BACKGROUND: H3 K27M-mutant diffuse midline gliomas (DMGs) are highly aggressive malignancies of the central nervous system that affect both pediatric and adult populations. The immune layout and genetic changes within the tumor microenvironment associated with these high-grade malignancies are thought to play an integral role in the phenotypic differences in tumor presentation and clinical course between both populations. Comparative immune landscape between pediatric and adult DMGs is not known. METHODS: The NanoString GeoMxTM Digital Spatial Profiler platform was used to determine the immune marker and genetic layout in a cohort of both pediatric and adult H3 K27M-mutant DMG tissue samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to identify and separate out the various components within the tumor tissues from selected regions of interest. The resultant information was then pooled into libraries that were run through the Illumina sequencing system to assess gene expression and proteomics for both cohorts of samples. RESULTS: Our data revealed that there were immune and genetic marker changes that were seen within both populations across multiple regions of interest within the tumor tissues. A number of immune cell types and protein markers exhibited significant difference between the pediatric and adult tissue cohorts. CONCLUSION: The digital spatial analysis of pediatric and adult H3 K27M-mutant DMGs show different immune and genetic profiles which are proposed to contribute to the clinical differences between both populations. These observed differences may play a role in future treatments for H3 K27M-mutant DMGs and further research is needed to explore this.

Evaluating anticancer agents on 3D bioprinted organoid tumors (BOT) to reduce cost and accelerate therapeutic discovery.

e13500 Background: Despite recent advances in therapeutics, cancer remains the second leading cause of death worldwide. Next generation cancer models hold the promise of breaking this stranglehold. However, extracting adequate tissue for precision and personalized medicine ex vivo models can be difficult depending on tumor type and tissue site. Mechanisms to expand tissue include patient derived xenografts and patient derived organoids. The former imposes a large time window to establish while the latter is constrained in space by sizescales. Advances in 3D bioprinting have improved the ability to reproduce the three-dimensionality and heterogeneity of the tumor microenvironment. Here we present a novel mechanism to address space and time constrains with 3D bioprinted organoid tumors (BOT) that mimic core needle biopsy tissue to reduce both cost and time to market for novel therapeutics making effective cancer therapy more accessible and equitable. The aim of this study is to produce and use BOT core biopsy tissue in ex vivo precision and personalized medicine applications. Methods: Bioinks prepared with MDA-MB-231 cells, fibrinogen, and thrombin will be deposited layer-by-layer on microporous substrate, in various geometrical configurations, and cured in stages to allow cells and matrix to self-assemble with limited degrees of freedom. Bioprinted organoid tissue will be fully cured to mimic patient biopsy cores, which will then be loaded in ex vivo bioreactors to evaluate sensitivity and resistance of small molecule and biologics. Results: Tissue 3D microarchitecture was validated using high-content fluorescence imaging and custom image processing applications. Diffusion of mock agents, small molecule, and nucleic acid stains was measured 200 mm deep in tissue. Differential activity in spatially distinct regions of intact BOT cores was quantified using advanced image processing modules. Conclusions: Patient derived BOT cores could be used both as a predictive model to screen drugs on an individualized basis and to uncover new therapeutic targets to improve efficacy and reduce toxicity. Due to their ability to replicate the physical and biochemical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine models can provide more accurate data on drug efficacy and toxicology when compared to in vitro cancer models.

Evaluating efficacy on tumor biopsy tissue ex vivo before treatment for equitable cancer care.

e13502 Background: Nearly half the world will be diagnosed with cancer. Each year, > 1.7 million new cancers are diagnosed in the US. Worse, systemic therapy turns out to be ineffective in 70% of patients because those drugs do not match the patient’s tumor, imposing a substantial physical, emotional, and financial burden. Each tumor’s distinct composition and heterogeneity can result in diverse responses to the same treatment. There is a critical and urgent need to develop ex vivo personalized cancer models that match patients with the right treatment – before treatment. Here we present a low-cost ex vivo personalized cancer model to rapidly evaluate effectiveness of various therapeutics on intact core biopsy tissue obtained from a patient’s tumor. Objective: The goal of this study is to deliver multiple isolated fluid streams to spatially distinct regions (SDRs) of intact core biopsy specimens for evaluating efficacy of multiple anticancer agents simultaneously. Methods: Core biopsies were generated from xenograft and resected human tumor tissue using 18-gauge and 20-gauge spring-loaded biopsy systems. Xenografts were established using cell lines with known sensitivity and resistance profiles. Fluid streams with anticancer agents, nucleic acid stains, and mock drugs were delivered to spatially distinct regions (SDRs) on intact core biopsy specimens in our patented Personalized Oncology Efficacy Test (POET) lab-chip bioreactors. High content imaging and custom image processing algorithms were used to measure differential activity. Results: No lateral fluid surface conduction, and only minimal lateral diffusion, was observed up to 200 μm deep in tissue. Extent of observed lateral diffusion was within operational parameters of the bioreactor. Integrity of spatially distinct regions on intact core tissue was maintained. Differential activity from anticancer agents compared to mock drugs was observed and quantitatively measured using custom image processing algorithms. Conclusions: The NCI warns that “current methods to assess potential cancer treatments are cumbersome, expensive, and often inaccurate.” A personalized medicine approach, based on efficacy observed on tissue directly from the patient’s own tumor, is fundamentally better than the current approach. Low-cost next generation ex vivo cancer models that can rapidly match treatments to tumors, such as POET, could transform cancer treatment and make a significant impact on the lives of patients.

Interactions and spectroscopic characteristics of propidium dication on soluble graphene oxides

Propidium is widely used as a red-fluorescent nucleic acid stain and universal cell death indicator, and recently experimental understanding of the electron transfer and fluorescence quenching of adsorbed fluorophores on carbon surfaces has attracted intense attention. In this work, the soluble graphene fragments were predigested as coronene oxides, and the aromaticity of coronene, a coronene oxide GO and a coronene oxide dianion GO2− was measured by HOMA indices and visualized with LOL-π maps by the M06-2X method. The weak interactions and π-π stacking between propidium and coronene, GO or GO2− in Complexes 1, 2 and 3 were confirmed by NCI analyses and frontier molecular orbitals. Then the absorption and fluorescence spectra were simulated by the TD-M06-2X method, and hole-electron analyses were employed to characterize electron excitations of these complexes. It is found that the absorption and fluorescence spectra of Complex 1 is much weaker than those of free propidium and Complex 2. The S1 → S0 transitions (LUMO→HOMO, π*→π) in the fluorescent spectra denote that these complexes can emit fluorescence in the visible-light region.

Differentiation of DNA or membrane damage of the cells in disinfection by flow cytometry

Recently, the viabilities changes of fungal spores in the water supply system during different disinfection processes have been revealed. SYBR Green I (SG), a nucleic acid stain, its fluorescence intensity is correlated with the amount of double-stranded DNA. This study established a new method through successive SG-SG-PI staining (PI: Propidium Iodide) with flow cytometry (FCM). It could successfully distinguish DNA damage and membrane damage of fungal spores, clearly elucidating the intrinsic disinfection mechanism during the chemical disinfection. This method was briefly described as follows: firstly, (1) the fungal spores were stained with SG and washed by centrifugation; and then, (2) the washed spores were treated with disinfectants and terminated; after that, (3) the disinfected spores were re-stained with SG and analyzed by FCM; finally, (4) the SG re-stained spores were stained with PI and analyzed by FCM. The percentages of spores with DNA damage and membrane damage were determined by the fluorescence intensity obtained from steps (3) and (4), respectively. The repeatability and applicability of this developed method were confirmed. It was further applied to explore the inactivation mechanism during chlorine-based disinfection, and results demonstrated that chloramine attacked the DNA more seriously than the membrane, while chlorine and chlorine dioxide worked in a reverse way.

Plant Growth-Promoting Ability of Mycorrhizal Fusarium Strain KB-3 Enhanced by Its IAA Producing Endohyphal Bacterium, Klebsiella aerogenes.

Fusarium oxysporum KB-3 had been reported as a mycorrhizal fungus of Bletilla striata, which can promote the seed germination and vegetative growth. Endohyphal bacteria were demonstrated in the hyphae of the KB-3 by 16S rDNA PCR amplification and SYTO-9 fluorescent nucleic acid staining. A strain Klebsiella aerogenes KE-1 was isolated and identified based on the multilocus sequence analysis. The endohyphal bacterium was successfully removed from the wild strain KB-3 (KB-3−), and GFP-labeled KE-1 was also transferred to the cured strain KB-3− (KB-3+). The production of indole-3-acetic acid (IAA) in the culturing broths of strains of KE-1, KB-3, KB-3−, and KB-3+ was examined by HPLC. Their IAA productions were estimated using Salkowski colorimetric technique. The highest concentrations of IAA were 76.9 (at 48 h after inoculation), 31.4, 9.6, and 19.4 μg/ml (at 60 h after inoculation), respectively. Similarly, the three fungal cultural broths exhibited plant promoting abilities on the tomato root and stem growth. The results indicated that the ability of mycorrhizal Fusarium strain KB-3 to promote plant growth was enhanced because its endohyphal bacterium, Klebsiella aerogenes KE-1, produced a certain amount of IAA.

Quantitative Imaging of Bacteriophage Amplification for Rapid Detection of Bacteria in Model Foods.

Rapid detection of bacteria in water and food samples is a critical need. The current molecular methods like real-time PCR can provide rapid detection after initial enrichment. However, these methods require significant preparation steps, specialized facilities to reduce contamination, and relatively expensive reagents. This study evaluates a novel approach for detecting bacteria based on imaging of bacteriophage amplification upon infection of the target host bacteria to mitigate some of these constraints and improve the specificity of discriminating live vs. dead bacteria. Thus, this research leverages the natural ability of lytic bacteriophages to rapidly amplify their genetic material and generate progeny phages upon infecting the host bacterium. This study uses a nucleic acid staining dye, a conventional fluorescence microscope, and quantitative image analysis for imaging the amplification of bacteriophages. The sensitivity and assay time for imaging-based quantification of phage amplification for detecting Escherichia coli were compared with RT-PCR and the standard plaque-forming assay for detection phage amplification in model systems, including coconut water and spinach wash water. The results demonstrate that the imaging approach matches both the sensitivity and speed for detecting E. coli using the RT-PCR method without requiring isolation of nucleic acids, expensive reagents, and specialized facilities. The quantitative imaging results demonstrate the detection of 10 CFU/ml of E. coli in coconut water and simulated spinach wash water with a chemical oxygen demand (COD) of 3,000 ppm within 8 h, including initial enrichment of the bacteria. In summary, the results of this study illustrate a novel phage amplification-based approach for detecting target bacteria in complex food and water samples using simple sample preparation methods and low-cost reagents.

In Vitro Production of Neutrophils Extracellular Traps Is Affected by the Lactational Stage of Dairy Cows.

Simple SummaryPolymorphonuclear leukocytes (PMN) are the first line and most abundant immune cells against infection. They kill bacteria via phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). The formation of NETs is always accompanied by the production of reactive oxygen species (ROS). We aimed to research the NETs and ROS formation capacity of PMN in different lactational stages of Holstein cows. We also validated a model to mimic infection and inflammation to study the NETs and ROS formation capacity in vitro. Results show that the basal NETs and ROS values of PMN harvested from peripartum and lactating cows were higher than those in nulliparous heifers. The in vitro stimulation, using inflammatory products, of PMN derived from nulliparous heifers increased the production of ROS and NETs. PMN isolated from peripartum and lactating cows are primed to produce NETs and ROS, and this potentially contributes to the cows’ unfavorable inflammatory and immune status at these critical periods in their lactation cycle. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic and study fundamental aspects of the production of NETs and ROS in vitro.We aimed to research the neutrophil extracellular traps (NETs) and reactive oxygen species (ROS) formation capacity of polymorphonuclear cells (PMN) during different lactational stages of Holstein cows. We also aimed to validate a model which could mimic infection and inflammation in vitro by adding increasing concentrations of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) to PMN suspensions isolated from nulliparous heifers and evaluate their capacity to produce NETs and ROS. In 3 replicates, we collected blood from nulliparous heifers (n = 3), cows at the end of gestation (n = 3), early postpartum (n = 3) and in mid-lactation (n = 3) in which PMN were isolated. The production of ROS in PMN were assessed using the 2’,7’-Dichlorofluorescein diacetate method, while the SYTOX Orange and Quant-iT™ PicoGreen dsDNA ultra-sensitive nucleic fluorescent acid staining methods were applied in order to quantitatively analyze the formation of NETs. Statistical analyses were performed via linear regression models using the replicate as a random. ROS values of PMN harvested from peripartum cows were 1.3 times increased compared with those in nulliparous heifers (p < 0.01). Compared with nulliparous heifers, the production of NETs by PMN isolated from mid-lactation and postpartum cows was 2.1 and 2.5 times higher (p < 0.01), respectively. In 3 replicates, in vitro stimulation of PMN isolated from nulliparous heifers (n = 3) with LPS linearly increased the production of ROS and NETs (R2 = 0.96 and 0.86, respectively). Similarly, when PMN isolated from nulliparous heifers were stimulated with PMA, a linear increase in the production of ROS (R2 = 0.99) and NETs (R2 = 0.78) was observed. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic inflammation and study fundamental aspects of the production of NETs and ROS in vitro.

NETosis in Long-Term Type 1 Diabetes Mellitus and Its Link to Coronary Artery Disease.

BackgroundNeutrophil extracellular traps NETs have been linked to glucose and the pathogenesis of type 1 diabetes mellitus (T1DM). NETs also play a role in vascular inflammation and the development of coronary artery disease (CAD). The role of NETs in CAD progression in patients with long-term T1DM is unclear. We aimed to 1) investigate whether levels of circulating NETs markers were elevated in long-term T1DM subjects compared to controls, and 2) explore whether levels of NETs were related to the presence of CAD.Material and Methods102 patients with > 45 years of T1DM and 75 age-matched controls were enrolled in a cross-sectional study. Median age was 62 years. Computed tomography coronary angiography (CTCA) was performed in 148 subjects without established coronary heart disease. For the current study, CAD was defined as a coronary artery stenosis >50%. Double-stranded deoxyribonucleic acid (dsDNA) was measured by a nucleic acid stain, myeloperoxidase-DNA (MPO-DNA), citrullinated histone 3 (H3Cit) and peptidylarginine deiminase 4 (PAD4) by ELISAs, while gene expression of PAD4 was measured in leukocytes from PAXgene tubes.ResultsCirculating MPO-DNA levels were significantly lower in patients with T1DM than in controls (0.17 vs 0.29 OD, p<0.001), while dsDNA, H3Cit, PAD4 and gene expression of PAD4 did not differ with respect to the presence of T1DM. There were no significant associations between NETs markers and HbA1c in the T1DM group. None of the NETs markers differed according to the presence of CAD in patients with T1DM. While all circulating NETs markers correlated significantly with circulating neutrophils in the control group (r=0.292-393, p<0.014), only H3Cit and PAD4 correlated with neutrophils in the T1DM group (r= 0.330-0.449, p ≤ 0.001).ConclusionsIn this cross-sectional study of patients with long-term T1DM and age-matched controls, circulating NETs levels were not consistently associated with the presence of T1DM or glycemic status, and did not differ according to the presence of CAD in patients with T1DM. Our results entail the possibility of altered neutrophil function and reduced NETosis in T1DM. This warrants further investigation.

Bacterial Dye Release Measures in Response to Antimicrobial Peptides.

Assessment of bacterial dye release following exposure to antimicrobial peptides (AMPs) provides a detailed understanding regarding their interaction with the inner and outer membrane of bacteria, and the leak of bacterial intracellular materials. This underpins the overall antimicrobial mechanism of these membrane-active peptides. DiSC3(5) is a membrane potential sensitive dye and can characterize the changes in bacterial membrane potential following exposure to AMPs (see Note 1). SYTOX Green is a nucleic acid stain that enters the cell upon loss of membrane integrity after exposure to AMPs and binds to DNA. SYTO9 is another nucleic acid stain, whereas propidium iodide (PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Both of these stains are widely used to monitor the viability of bacteria following exposure to AMPs. This chapter describes the methods of using these as bacterial dye release experiments for assessment of the antimicrobial mechanism of AMPs.

Dodecylamine rapidly kills of spores of multiple Firmicute species: properties of the killed spores and the mechanism of the killing.

Previous work showed that Bacillus subtilis dormant spore killing and germination by dodecylamine take place by different mechanisms. This new work aimed to optimize killing of B. subtilis and other Firmicutes spores and to determine the mechanism of the killing. Spores of seven Firmicute species were killed rapidly by dodecylamine under optimal conditions and more slowly by decylamine or tetradecylamine. The killed spores were not recovered by additions to recovery media, and some of the killed spores subsequently germinated, all indicating that dodecylamine-killed spores truly are dead. Spores of two species treated with dodecylamine were more sensitive to killing by a subsequent heat treatment, and spore killing of at least one species was faster with chemically decoated spores. The cores of dodecylamine-killed spores were stained by the nucleic acid stain propidium iodide, and dodecylamine-killed wild-type and germination-deficient spores released their stores of phosphate-containing small molecules. This work indicates that dodecylamine is likely a universal sporicide for Firmicute species, and it kills spores by damaging their inner membrane, with attendant loss of this membrane as a permeability barrier. There is a significant need for agents that can effectively kill spores of a number of Firmicute species, especially in wide area decontamination. Dodecylamine appears to be a universal sporicide with a novel mechanism of action, and this or some comparable molecule could be useful in wide area spore decontamination.

Complement activation is associated with neutrophil extracellular traps and all-cause mortality in ST-elevation myocardial infarction

Abstract Background The complement system and neutrophil extracellular traps (NETs) are both parts of the innate immune system, and have been implicated in the ischemia-reperfusion injury in patients with ST-elevation myocardial infarction (STEMI). There is experimental evidence of reciprocal activation between the complement system and NETs. Any such link in patients with STEMI has not been investigated. Purpose To investigate a potential association between complement activation and clinical outcomes after STEMI, and assess any interplay between complement activation and NETs in this situation. Methods Patients with ST-elevation myocardial infarction were included at a median of 18 hours after percutaneous coronary intervention (n=864). The terminal complement complex (TCC) was measured by ELISA as a marker of complement activation. As markers of NETs were myeloperoxidase-deoxynucleic acid (MPO-DNA) and citrullinated histone 3 (CitH3) measured by ELISAs, while double stranded DNA (dsDNA) was measured by a nucleic acid stain. Patients were followed for a median of 4.6 years. The primary endpoint was a composite of new myocardial infarction, unscheduled revascularization, stroke, hospitalization for heart failure and death, whichever occurred first. All-cause mortality was also recorded. Results The composite endpoint occurred in 184 (21.3%) patients, while 70 (8.1%) died during follow-up. When dichotomizing at median TCC, the group with above-median TCC levels did not have an increased risk of reaching the composite endpoint (hazard ratio (HR): 1.069, 95% CI: [0.801, 1.428], p=0.651). However, this group exhibited an increased risk of all-cause mortality (HR: 1.650, 95% CI: [1.020, 2.671], p=0.041). This risk persisted when adjusting for age, sex, hypertension and LDL-cholesterol (HR: 1.673, 95% CI: [1.014, 2.761], p=0.044), but the significance was lost when adjusting for NT-proBNP (HR: 1.492, 95% CI: [0.885, 2.515], p=0.133). TCC was correlated to dsDNA (r=0.127, p&amp;lt;0.001) and CitH3 (r=0.102, p=0.003), but not MPO-DNA. The group with both TCC and dsDNA in the highest quartile exhibited a significantly higher incidence of all-cause mortality than the remaining population (17.6% vs, 7.2%, p=0.002). When examining the predictive value of TCC and dsDNA on all-cause mortality in ROC curve analysis, the area under the curve (AUC) for TCC was 0.549 (95% CI: [0.472, 0.625]), while the AUC for dsDNA was 0.653 (95% CI: [0.584, 0.722]). When combining TCC and dsDNA the predictive value was marginally higher than for TCC alone (AUC: 0.649, 95% CI: [0.579, 0.720]) Conclusion In this STEMI population, complement activation measured by TCC was not associated with the primary composite endpoint, but was associated with increased risk of death. TCC was weakly correlated with markers of NETs. Despite a high mortality rate in patients with high levels of TCC and dsDNA, combining these variables did not increase the prognostic value compared to TCC alone. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Stein Erik Hagen's Foundation for Clinical Heart Research Survival according to cox regression

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Towards computer aided diagnosis of infective endocarditis in whole-slide images of heart valve tissue using FISH

Abstract Infective endocarditis (IE) is an infection of the endocardium, and the heart valves associated with high morbidity and mortality. Fluorescence in situ Hybridization (FISH) is a molecular imaging technique used for diagnosis of IE based on histological heart valve tissue sections. FISH allows detection and identification of microorganisms and gives information about their quantity and spatial distribution. This information is important to guide appropriate antibiotic treatment. However, as manual FISH image analysis is time- and costexpensive, an automated image analysis pipeline (consisting of tissue segmentation, bacteria detection, and spot detection modules) is proposed to assist locating potential regions with microorganisms. The proposed approach was evaluated in a study, where five observers manually assessed a set of 171 fields-of-view (FoVs) captured in 400-fold magnification from 10 randomly chosen WSI for the presence of microorganisms, morphologically detected by the nucleic acid stain DAPI. The task of the observers was to mark the presented image using a 2-class score (‘positive/questionable’ or ‘negative’). The human assessment was compared to the results suggested by the algorithm. The proposed algorithm locates and ranks potential regions with microorganisms in heart valve sections so that experts can validate them in higher power FoVs for the presence of bacteria and identify their species. The automated system for preselecting and recommending adequate FoVs is thus a starting point to support experts and save human resources. It is now ready to be further developed for the detection of bacteria by FISH.

The FcγRIII Engagement Augments PMA-Stimulated Neutrophil Extracellular Traps (NETs) Formation by Granulocytes Partially via Cross-Talk between Syk-ERK-NF-κB and PKC-ROS Signaling Pathways.

Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cell in the circulation capable of neutrophil extracellular traps (NETs) formation after stimulation. Both NADPH oxidase-dependent and -independent pathways are involved in NET formation. The IgG is the most abundant immunoglobulin in human serum. However, the impact of the circulating IgG on NET formation is totally unexplored. In this study, the all-trans retinoic acid (ATRA)-induced mature granulocytes (dHL-60) were pre-treated with monomeric human IgG, papain-digested Fab fragment, crystallizable IgG Fc portion, rituximab (a human IgG1), or IgG2. The NET formation of the dHL-60 in the presence/absence of phorbol 12-myristate 13-acetate (PMA) stimulation was then measured by the fluorescent area after SYTOX green nucleic acid stain. The intracellular reactive oxygen species (ROS) generation was measured by flow cytometry. Total and phosphorylated Syk, SHP-1, and ERK were detected by immunoblot. We found that human monomeric IgG and its subclasses IgG1 and IgG2 per se induced negligible NET formation of dHL-60, but the FcγRIII engagement by these IgG subclasses and Fc portion augment PMA-stimulated dHL-60 NET formation in a dose-dependent manner. Furthermore, we found that increased Syk and ERK phosphorylation, intracellular ROS generation, and pro-inflammatory cytokines, IL-8 and TNF-α, production could be induced after FcγRIII engagement. Blocking FcγRIII engagement by a specific antibody diminished the augmented NET formation. In conclusion, we discovered that cross-talk between FcγRIII engagement-induced Syk-ERK and PMA-induced PKC signaling pathways augment NET formation of dHL-60 via increased ROS generation and pro-inflammatory cytokines, IL-8 and TNF-α, production.

Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein.One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

In silico recombinant plasmid design of pHA171 with phdABCD insertion for ethidium bromide degradation

Background: Ethidium bromide is a common reagent that is used in nucleic acid staining. However, ethidium bromide has toxic and carcinogenic properties that are harmful to the environment. Phenanthrene dioxygenase (encoded by phdA, phdB, phdC, and phdD genes) in Nocardioides sp. KP7 can oxidize the phenanthridine structure aim to eliminate carcinogenic properties.&#x0D; Objective: This study aims to visualize and predict the structure, active site, and characteristics of the phenanthrene dioxygenase using bioinformatics tools.&#x0D; Methods: Plasmid design were prepared by inserting genes of interest phdA, phdB, phdC, and phdD from the NCBI database. Furthermore, several protein analysis tools were used for structure visualization, active site enzyme improvement, and protein characteristic of phenanthrene dioxygenase.&#x0D; Results: The prediction results found that phenanthrene dioxygenase reacts with the ethidium bromide substrate through the interaction of Fe3+ ions with water. The solubility level of phenanthrene dioxygenase protein is 0.404, suggesting that the protein has low solubility. The protein isoelectric point (pI) is between 5.17 to 5.36, and the protein molecular weight is 121.143 kDa.&#x0D; Conclusion: In silico analysis has supported that recombinant plasmid met characteristics for the construct which consists of gene interest and protein library.

Flow Cytometric Quantification of Cytotoxic Activity in Whole Blood Samples.

Current methods for the determination of cell-mediated cytotoxic activity in blood samples usually isolate peripheral blood mononuclear cells by density gradient centrifugation or alternatively use erythrocyte lysis. Both centrifugation and red cell lysis can cause cellular depletion and cell dysfunction, resulting in erroneous measurements. To address limitations of current assays, we developed an improved strategy to determine cellular cytotoxicity using flow cytometry. Viable nucleic acid stains are used to identify live nucleated cells and discriminate them from non-nucleated erythrocytes, platelets, and debris while avoiding lysing and washing steps to maintain cell functionality. To detect target cells, we have used two different labeling approaches. In the first approach, EGFP-labeled K562 human chronic myelogenous leukemia cells provide a "ready-to-use" target without the need of additional for labeling or staining. For the second approach, we perform parallel cytotoxicity assays in the presence of wild-type K562 cells previously loaded with a fluorescent dye that has spectral properties similar to those of EGFP. Given the importance of cytotoxic assays and the deleterious effects of current sample preparation methods, the aim of this study was to adapt this "untouched cells" flow cytometry method to study cytotoxic activity using unlysed whole blood samples and fluorescent target cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for cell-mediated cytotoxic activity determination in unlysed whole blood Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of cell-mediated cytotoxic activity using unlysed whole blood samples Support Protocol 1: Optimization of the performance of target cell labeling approaches Support Protocol 2: Assessment of the linearity and reproducibility of cytotoxicity assays.

FLOW CYTOMETRIC AND MOLECULAR ANALYSIS OF POSSIBLE PROTOZOAL CONTAMINATION OF DRINKING WATER IN TANTA, EGYPT

Protozoal water contamination is an alarming cause of countless waterborne outbreaks. The mosteminent causal protozoa are Cryptosporidium and Giardia species (spp.) as they can endure aquaticenvironment even with chlorine disinfectants. The currently used traditional techniques cannot permitan easy detection of the waterborne protozoa concerning their count, viability, and pathogenicity. Thepresent work detected protozoal contamination of the drinking water in Egypt with the determinationof their load, viability and potential pathogenicity. Four techniques were compared including conventionalstaining techniques, immunofluorescence (IF) staining, flow cytometry (FC) and molecularstudy. Also, viability was assessed by conventional trypan blue stain and nucleic acid stain. Along ayear, 64 water samples were collected and concentrated from water tanks and tap water of differentdistricts, significant differences (P < 0.001) was between the different techniques in each season regardingthe detection of Giardia cysts and Cryptosporidium oocysts. Number of total positive sampleswas significantly higher in tank water than tap water (P < 0.001) especially at summer. Flow cytometryand nested polymerase chain reaction (nPCR) proved to be much more sensitive than IF assay, andconventional staining techniques. Regarding viability, nucleic acid stain was more sensitive than trypanblue stain (P < 0.001). Also, C. parvum predominate other Cryptosporidium genotypes.