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Accurate airborne virus monitoring is important for preventing the spread of infectious diseases. Although standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) can efficiently detect viral ribonucleic acid (RNA), it cannot determine whether the RNA is associated with active (infectious) or inactive (non-infectious) viruses. Plaque assay is the gold standard for determining viral infectivity but is laborious and time-consuming. This study explored the viral infectivity of H1N1 influenza virus and human coronavirus (HCoV-229E) using capsid integrity RT-qPCR, where virus samples were pretreated with reagents penetrating viruses with damaged capsids, impeding amplification by binding to their RNA. Therefore, the amplified signals corresponded solely to active viruses with undamaged capsids. Propidium monoazide (PMA) and platinum (IV) chloride (PtCl 4 ) were used to investigate the effects of reagent concentration. Feasibility tests revealed that PtCl 4 was more efficient than PMA, with optimal concentrations of 125-250 μM and 250-500 μM for H1N1 influenza virus and HCoV-229E, respectively. The results of percentage of active virus showed that capsid integrity RT-qPCR provided a trend similar to that of plaque assay, indicating an accurate measure of viral infectivity. Virus sampling in the laboratory and field highlighted the precision of this methodology for determining viral infectivity. Therefore, this methodology enables rapid and accurate detection of infectious airborne H1N1 influenza virus and HCoV-229E, allowing swift response to outbreaks., 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 Elsevier B.V. All rights reserved.)

Dish sponges are known to support the proliferation of human bacterial pathogens, yet they are commonly used by consumers. Exposure to foodborne pathogens via sponge use may lead to illness, a serious concern among susceptible populations. The extent of exposure risks from sponge use has been limited by constraints associated with culture-independent or dependent methods for bacterial community characterization. Therefore, five used dish sponges were characterized to evaluate the presence of viable bacterial foodborne pathogens using the novel application of propidium monoazide (PMA) treatment and targeted 16S rRNA gene amplicon sequencing. Select pathogen viability was confirmed using targeted selective enrichment. The taxonomic abundance profiles of total and viable sponge microbiomes did not vary significantly. The numbers of unique bacterial species (p = 0.0465) and foodborne pathogens (p = 0.0102) identified were significantly lower in viable sponge microbiomes. Twenty unique bacterial foodborne pathogens were detected across total and viable sponge microbiomes, and three to six viable foodborne pathogens were identified in each sponge. Escherichia coli and Staphylococcus aureus were identified in each viable sponge microbiome, and viable E. coli were recovered from two sponges via targeted selective enrichment. These findings suggest that sponge-associated bacterial communities are primarily viable and contain multiple viable bacterial foodborne pathogens., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot of stone fruits and almond (Prunus spp). Detection of Xap is typically carried out using quantitative real-time PCR (qPCR) combined with culture-based isolation. However, qPCR does not differentiate between viable and dead cells, potentially leading to an overestimation of the infective population in a sample. Such overestimation could result in unnecessary phytosanitary measures. The present study aims to develop a specific protocol ideally targeting to detection of only live Xap bacterial cells. To address this challenge, the viable quantitative PCR (v-qPCR) method was evaluated using three nucleic acid-binding dyes: propidium monoazide (PMA), a combination of PMA and ethidium monoazide (EMA), and PMAxx™, an improved version of PMA. PMAxx™ proved to be the most suitable dye for the detection and quantification of living bacterial cells. This methodology was also evaluated in infected plant material over time and can be considered a rapid and reliable alternative to PCR methods for detecting only those putative infective Xap that may pose a risk for Prunus crops. KEY POINTS: • Protocol to detect biofilm and planktonic viable X. arboricola pv. pruni cells. • Host validated protocol. • Benefits, reduction of chemicals in disease control., (© 2024. The Author(s).)

Escherichia coli O157:H7 is a major foodborne pathogen that poses a significant threat to food safety and human health. Rapid and sensitive detection of viable Escherichia coli O157:H7 can effectively prevent food poisoning. Here, we developed a microwell-confined and propidium monoazide-assisted digital CRISPR microfluidic platform for rapid and sensitive detection of viable Escherichia coli O157:H7 in food samples. The reaction time is significantly reduced by minimizing the microwell volume, yielding qualitative results in 5 min and absolute quantitative results in 15 min. With the assistance of propidium monoazide, this platform can eliminate the interference from 99% of dead Escherichia coli O157:H7. The direct lysis method obviates the need for a complex nucleic acid extraction process, offering a limit of detection of 3.6 × 10 1 CFU mL -1 within 30 min. Our results demonstrated that the platform provides a powerful tool for rapid detection of Escherichia coli O157:H7 and provides reliable guidance for food safety testing.

This study compares the plate count (PC) and the Propidium Monoazide-quantitative Polymerase Chain Reaction (PMA-qPCR) methods to assess the growth of a cocktail of three serotypes of Salmonella enterica (Heidelberg, Typhimurium, and Enteritidis) in cooked, sliced, and vacuum-packaged turkey breast (STB) under isothermal storage temperatures (8 °C-20 °C), using predictive models. Standard curves were developed for PMA-qPCR, demonstrating high efficiency (101%) and sensitivity, with quantification limits ranging from 1 to 2 log 10  CFU/g for all temperatures studied. Comparative analysis revealed a significant correlation (R 2  = 0.99; 95% CI) between the PC and PMA-qPCR methods; however, the agreement analysis indicated a mean difference (Bias) of -0.11 log 10  CFU/g (p < 0.05), suggesting underestimation by the PC method. This indicates the presence of stressed or viable but nonculturable (VBNC) cells, detectable by PMA-qPCR but not by PC. The Baranyi and Roberts model showed a good ability to describe the behavior of S. enterica cocktail in STB for PC and PMA-qPCR data under all isothermal conditions. The exponential secondary model more accurately represented the temperature dependence of the maximum specific growth rate compared to the Ratkowsky square root model, with R 2 values ≥ 0.984 and RMSE values ≤ 0.011 for both methods. These results suggest that combining PMA-qPCR with predictive modeling allows for a more accurate prediction of S. enterica growth, compared to PC method. In the event of cold chain disruptions of meat products, the use of PMA-qPCR method allow the quantification of VBNC cells, that can still pose a health risk to consumers, especially in ready-to-eat products., Competing Interests: Declaration of competing interest The authors declare that they have no financial or non-financial interests that could be perceived as potential conflicts of interest regarding this research. None of the authors have received any form of compensation, including honoraria or consultancy fees, from any organization that may gain or lose financially from the publication of this manuscript. The authors have no financial or personal relationships that could bias this work or create a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aimed to compare the performance of flow cytometry methods with plate counting for the enumeration of bacteria, using Bacillus cereus as a model organism. It was found that the cFDA-propidium iodide, CellROX™ Green-propidium iodide, and DiOC 2 dye techniques had similar accuracy to plate counting, while the SYTO 24-propidium iodide dye technique was not as accurate. The four dye techniques had comparable precision to plate counting, with the CellROX™ Green-propidium iodide dye having the greatest precision. The consistency of the position and shape of the cell clusters on the flow cytometry plots, and the extent of separation of the cell from background clusters, was greatest with the DiOC 2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC 2 and CellROX™ Green-propidium iodide dyes performed well, even when a sample was measured containing reconstituted whole milk powder at a 10 -1 dilution, without the use of sample preparation to specifically remove the milk constituents prior to measurement. Given gating of only one cell cluster was required to be managed with the DiOC 2 dye, to determine the viable number of cells, it was found that the DiOC 2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC 2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10 -8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Bacterial spot is one of the most serious diseases of peach caused by the pathogen Xanthomonas arboricola pv. pruni (XAP), leading to early defoliation and unmarketable fruit. The pathogen can overwinter in peach twigs and form spring cankers, which are considered the primary inoculum source for early season leaf and fruitlet infection. The amount of overwintering bacterial inoculum plays a critical role for the bacterial spot development, but no reliable quantification method is available. Thus, we developed a long-amplicon propidium monoazide (PMA)-quantitative PCR (qPCR) assay for specific detection of viable XAP cells. The optimized PMA-qPCR assay used 20 μM of PMAxx for pure bacterial suspensions and 100 μM for peach twig tissues. The Qiagen Plant Pro Kit with an additional lysozyme digestion step was the DNA extraction protocol that yielded the best detection sensitivity with the bacteria-spiked peach twig extracts. The PMA-qPCR assay was tested with different mixtures of viable and heat-killed XAP cells in pure bacterial suspensions and bacteria-spiked peach twig tissues. The results showed that this assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 10 3 CFU/ml with the presence of up to 10 7 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. For mixtures of viable and dead cells, the PMA-qPCR results were linearly correlated with the predicted concentrations of viable XAP ( R 2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees., Competing Interests: The author(s) declare no conflict of interest.

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris-Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non-CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability-DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram + , Gram - ) and polybacterial cultures. Any of the three methods can be used and adapted to the needs of each project and allow users to adapt the combination according to the configuration of their cytometer. Having been tested on six bacterial populations, validated on two cytometers, and repeated at least two times in each evaluated condition, we consider this method reliable in the context of these conditions. The reliability of the results obtained in flow cytometry was successfully validated by applying this protocol to confocal microscopy, permeabilization, and also to follow cultures over time. This flow cytometry protocol for measuring bacterial viability under safer conditions also opens the prospect of its use for further bacterial characterization., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Squaraine dyes are a family of compounds known for their relevant photophysical and photochemical properties potentially useful as photosensitizing agents. Since pyridines have been introduced into the skeleton of several families of compounds to enhance their pharmacological activity, and this approach had not yet been performed on squaraines, novel dyes derived from benz[e]indole functionalized with picolyl- and dipicolylamine and N-ethyl and -hexyl chains were designed and synthesized. After being fully characterized, their interaction with human albumin was in vitro and in silico evaluated. Dyes were further assessed for their phototoxicity activity, and the most interesting ones were studied regarding cell localization and induction of morphological cell changes, genotoxicity, apoptosis and cell cycle arrest. The molecules with N-ethyl chains showed the greatest in vitro light-dependent cytotoxic effects, particularly the zwitterionic squaraine dye and the one bearing a single pyridine unit, which also exhibited a more significant interaction with human albumin. Phenotypically, the cells incubated with these squaraines became smaller and rounded after irradiation, the effects varying with the tested concentration. Genotoxic effects were observed even without irradiation, being more evident for the N-ethyl picolylamine-derived dye. The fluorescence emitted by Rhodamine 123 largely coincided with that emitted by the dyes, suggesting that they are found preferentially in mitochondria. After irradiation, an increase in the subG1 population was verified by propidium iodide-staining analysis by flow cytometry, indicative of cell death by apoptosis., 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 © 2021 Elsevier Masson SAS. All rights reserved.)

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log 10 copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced β-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Background: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells., Methods: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence., Results: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945., Conclusions: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

Live imaging allows observations of cell structures and processes in real time, to monitor dynamic changes within living organisms compared to fixed organisms. Fluorescence microscopy was used to monitor the dynamic infection process of the nematode parasitic bacterium Pasteuria sp. and the sugarcane root-lesion nematode, Pratylenchus zeae. Under fluorescence microscopy, green-autofluorescent globules were observed in live control and Pasteuria sp.-infected nematodes. Only nematodes killed by Pasteuria sp. or heat treated displayed a diffuse pattern of autofluorescence. Propidium iodide (PI), used as a cell membrane integrity indicator, confirmed that the nematode's cuticle acts as an impermeable barrier. PI stained cells/DNA of heat-treated control and Pasteuria sp.-infected P. zeae. PI as a counterstain facilitated the location of Pasteuria endospores on the cuticle surface of P. zeae. No PI staining was observed in sporangia and in endospores within the nematode body. However, PI specifically stained endospores on the cuticle surface and within the cuticle carcass showing, in mature propagules, a ring-like pattern. Live imaging, combined with fluorescence microscopy and fluorescent dyes such as PI, appears useful in live studies on plant nematode interactions with nematophagous bacteria.

Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca 2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Multiparametric flow cytometry is a technique utilized in translational experiments that utilizes fluorescently tagged antibodies and functional fluorescent dyes to measure proteins on the surface or in the cytoplasm of cells and to measure processes occurring within cells themselves. These fluorescent molecules, or fluorophores, can be tagged to antibodies to measure specific biological molecules such as proteins inside or on the surface of cells. Small organic compounds such as the nucleic acid binding dye propidium iodide (PI) can permeate compromised cell membranes when cells are no longer viable or used to measure DNA content of cycling cells. Successful completion of flow cytometry experiments requires expertise in both the preparation of the samples, acquisition of the samples on instruments, and analyses of the results. This chapter describes the principles needed to conduct a successful multiparameter flow cytometry experiment needed for drug development with references to well established internet resources that are useful to those less experienced in the field. In addition, we provide a brief introduction to data analysis including complex analysis of 10+ parameters simultaneously. These high-dimensional datasets require novel methods for analysis due to the volume of data collected, which are also introduced in this chapter.

Eosinophil apoptosis (programmed cell death) plays an important role in several inflammatory and allergic conditions. Apoptosis triggers various mechanisms including activation of cysteine-aspartic proteases (caspases) and is characterized by morphological and biochemical changes. These include cellular condensation, nuclear fragmentation, increased mitochondrial permeability with loss of membrane potential, and exposure of phosphatidylserine on the cell membrane. A greater understanding of apoptotic mechanisms, subsequent phagocytosis (efferocytosis), and regulation of these processes is critical to understanding disease pathogenesis and development of potential novel therapeutic agents. Release of soluble factors and alterations to surface marker expression by eosinophils undergoing apoptosis aid them in signaling their presence to the immediate environment, and their subsequent recognition by phagocytic cells such as macrophages. Uptake of apoptotic cells usually suppresses inflammation by restricting proinflammatory responses and promoting anti-inflammatory and tissue repair responses. This, in turn, promotes resolution of inflammation. Defects in the apoptotic or efferocytosis mechanisms perpetuate inflammation, resulting in chronic inflammation and enhanced disease severity. This can be due to increased eosinophil life span or cell necrosis characterized by loss of cell membrane integrity and release of toxic intracellular mediators. In this chapter, we detail some of the key assays that are used to assess eosinophil apoptosis, as well as the intracellular signaling pathways involved and phagocytic clearance of these cells.

New hybrids of 4-amino-2,3-polymethylenequinoline with different sizes of the aliphatic ring linked to butylated hydroxytoluene (BHT) by enaminoalkyl ( 7 ) or aminoalkyl ( 8 ) spacers were synthesized as potential multifunctional agents for Alzheimer's disease (AD) treatment. All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. Lead compound 8c , 2,6-di- tert -butyl-4-{[2-(7,8,9,10- tetrahydro-6H-cyclohepta[b]quinolin-11-ylamino)-ethylimino]-methyl}-phenol exhibited an IC 50 (AChE) = 1.90 ± 0.16 µM, IC 50 (BChE) = 0.084 ± 0.008 µM, and 13.6 ± 1.2% propidium displacement at 20 μM. Compounds possessed low activity against carboxylesterase, indicating likely absence of clinically unwanted drug-drug interactions. Kinetics were consistent with mixed-type reversible inhibition of both cholinesterases. Docking indicated binding to catalytic and peripheral AChE sites; peripheral site binding along with propidium displacement suggest the potential of the hybrids to block AChE-induced β-amyloid aggregation, a disease-modifying effect. Compounds demonstrated high antioxidant activity in ABTS and FRAP assays as well as inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Conjugates 8 with amine-containing spacers were better antioxidants than those with enamine spacers 7 . Computational ADMET profiles for all compounds predicted good blood-brain barrier distribution (permeability), good intestinal absorption, and medium cardiac toxicity risk. Overall, based on their favorable pharmacological and ADMET profiles, conjugates 8 appear promising as candidates for AD therapeutics.

We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Songfeng Chen et al.)

The cell membrane permeabilization in electroporation studies is usually quantified using fluorescent markers such as propidium iodide (PI) or YO-PRO, while Chinese Hamster Ovary cell line frequently serves as a model. In this work, as an alternative, we propose a sensitive methodology for detection and analysis of electroporation phenomenon based on bioluminescence. Luminescent mice myeloma SP2/0 cells (transfected using Luciferase-pcDNA3 plasmid) were used as a cell model. Electroporation has been studied using the 0.1-5 μs × 250 and 100 μs × 1-8 pulsing protocols in 1-2.5 kV/cm PEF range. It was shown that the bioluminescence response is dependent on the cell permeabilization state and can be effectively used to detect even weak permeabilization. During saturated permeabilization the methodology accurately predicts the losses of cell viability due to irreversible electroporation. The results have been superpositioned with permeabilization and pore resealing (1 h post-treatment) data using PI. Also, the viability of the cells was evaluated. Lastly, the SP2/0 tumors have been developed in BALB/C mice and the methodology has been tested in vivo using electrochemotherapy with bleomycin., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A new type of ultrathin fiber microprobe for selective electroporation is reported. The microprobe is 10 cm long and has a diameter of 350 µm. This microprobe is a low cost tool, which allows electroporation of an arbitrary selected single cell or groups of cells among population with use of a standard microscope and cell culture plates. The microprobe in its basic form contains two metal microelectrodes made of a silver-copper alloy, running along the fiber, each with a diameter of 23 µm. The probe was tested in vitro on a population of normal and cancer cells. Successful targeted electroporation was observed by means of accumulation of trypan blue (TB) dye marker in the cell. The electroporation phenomenon was also verified with propidium iodide and AnnexinV in fluorescent microscopy., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Ryszard Buczyński, Dariusz Pysz and Ryszard Stępień are inventors of patents owned by Institute of Electronic Materials Technology in Warsaw in technical field of the publication]., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Rapid and sensitive detection of live/infectious foodborne pathogens is urgently needed in order to prevent outbreaks and food recalls. This study aimed to (1) evaluate the incorporation of propidium monoazide (PMA) into PCR or LAMP assays to selectively detect viable Salmonella Enteritidis following sublethal heat or UV treatment, and autoclave sterilization; and (2) compare the detection of PMA-PCR and PMA-LAMP to DNA-based PCR and LAMP (without PMA), RNA-based RT-PCR and RT-LAMP, and culture-based methods. Nucleic acids (DNA or RNA) from 1-mL S. Enteritidis samples were used for PCR, RT-PCR, LAMP, and RT-LAMP assays. Serially diluted samples were plated on Xylose Lysine Tergitol-4 agar for cultural enumeration. Comparable detection of overnight cultured S. Enteritidis was obtained by PMA-PCR, PCR, and RT-PCR, though 1 to 2 log less sensitive than cultural assays. PMA-LAMP and RT-LAMP showed similar detection of overnight cultures, being 1 to 2 log less sensitive than the LAMP assay, and ∼4 log less than culture-based detection. Autoclaved S. Enteritidis did not test positive by RNA-based methods or PMA-PCR, but PMA-LAMP showed detection of 1 log CFU/mL. PMA-PCR and RT-PCR showed comparable detection of sublethal heat-treated cells to cultural assays, while PMA-LAMP showed 1 to 2 log less detection. Our results suggest that PMA-PCR and PMA-LAMP assays are not suitable for selective viable cell detection after UV treatment. While PMA-LAMP assay needs optimization, PMA-PCR shows promise for live/viable S. Enteritidis detection. PMA-PCR shows potential for routine testing in the food industry with results within 1-day, albeit depending on the inactivation method employed., (© 2020 Institute of Food Technologists®.)

Background: Mitochondrial dysfunction may relate to metabolic disorders. The relation between maternal and fetal mitochondrial function needs attention due to heritage. Objectives: To evaluate the use of the staining methods TetraMethylRhodamine Methyl Ester (TMRM) and Mitotracker Green (MTG) for flow cytometric measurements of umbilical cord blood mitochondrial function. Methods : 53 euthyroid at-term pregnant women and their offspring were included by blood collections. The offspring had blood drawn from the clamped umbilical cord. Flow cytometry with MTG, TMRM and Propidium Iodide were performed the following day. A cell count (antibody coating and flow cytometry) was performed for 9 maternal and cord samples. As a quality control, blood of 32 healthy donors was evaluated by flow cytometric analyzes same day as sampling and the following day to test stability of the measurements. Results: Cord mitochondrial measurements were lower than maternal. Maternal and cord mitochondrial function were positively correlated, especially reflected by MTG fluorescence-intensity (FI). Samples stored presented with very changed fluorescence patterns. However, the fluorescence intensity ratios MTG/TMRM of stained white blood cells were related within same day measurements, depicting an extensive and common bioenergetic cellular change. Conclusion: Cord blood flow cytometry by MTG- and TMRM- staining is possible with fluorescence intensity positively correlated to maternal fluorescence intensity. Storage of blood triggers mitochondrial dynamics. The methods are applicable with certain reservations, and they benefit from their non-invasive character compared to mitochondrial evaluation by muscle-biopsies.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

Fluorophores SYTO 9 and propidium iodide (PI) are extensively applied in medicine, food industry and environmental monitoring to assess the viability of bacteria. However, the actual performance of these dyes remains largely unknown. In addition, their effects on the physiology of cells have not been elucidated. Here we characterized the effects of these two dyes on Brevibacillus brevis under optimized staining. We found that SYTO 9 entered cells continuously while PI tended to adhere to the cell wall before entering the cell. In addition, results showed that a high amount of the dyes altered the physicochemical properties of membranes, improving their breakthrough. These results provide new perspectives and ideas for improving the characterization of bacterial viability using flow cytometry.

Aims: The aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species)., Methods and Results: Different materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs., Conclusions: The nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used., Significance and Impact of the Study: This study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4 × 10 3 , 4.8 × 10 3 , and 7.2 × 10 3  μJ/cm 2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8 × 10 3  μJ/cm 2 in 3 min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 10 0  CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4 h.

Poor control on the exfoliation of covalent organic frameworks (COFs) remains a disadvantage for their application as two-dimensional nanosheets. An equally important problem is the reversible control at the available surface charges on COFs. Herein, a strategy for the reversible exfoliation, re-stacking, and surface-charge control of a propidium iodide based ionic covalent organic framework, PI-TFP, using cucurbit[7]uril (CB[7]) induced molecular recognition, is reported. The surface charge on PI-TFP facilitates its initial self-exfoliation. However, complexation with CB[7] resulted in re-stacking with concomitant decrease in zeta potential from +28±3.0 to +0.004±0.003 mV. Addition of 1-adamantylamine hydrochloride (AD) facilitates decomplexation of PI-TFP from CB[7], resulting in exfoliation and an increase in zeta potential to +24±3.0 mV. Such control on the exfoliation, re-stacking, and the associated regulation of the surface charge in PI-TFP was exploited for controlling bacterial growth. Thus, the activity of E. coli and S. aureus bacteria obtained with the self-exfoliated PI-TFP could be reversibly controlled by the CB[7]/AD pair., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Use of anaerobic sludge digester is a common practice around the world for solids digestion and methane generation from municipal sewage sludge. Understanding microbial community structure is vital to get better insight into the anaerobic digestion process and to gain better process control. However, selective analysis of viable microorganisms is limited by DNA-based assays. In this study, propidium monoazide (PMA)-PCR with 16S rRNA gene sequencing analysis was used to distinguish live and dead microorganisms based on cell membrane integrity. Microbial community structures of PMA-treated and PMA-untreated anaerobic digester sludge samples were compared. Quantitative PCR revealed that 5-30% of the rRNA genes were derived from inactive or dead cells in anaerobic sludge digesters. This caused a significant decrease in the numbers of operational taxonomic units and Chao1 and Shannon indices compared with that of the PMA-untreated sludge. Microbial community analysis showed that majority of the viable microbiome consisted of Euryarchaeota, Bacteroidetes, Deltaproteobacteria, Chloroflexi, Firmicutes, WWE1, Spirochaetes, Synergistetes, and Caldiserica. On the other hand, after the PMA treatment, numbers of Alphaproteobacteria and Betaproteobacteria declined. These were considered residual microbial members. The network analysis also revealed a relationship among the OTUs belonging to WWE1 and Bacteroidales. PMA-PCR-based 16S rRNA gene sequencing analysis is an effective tool for uncovering viable microbiome in complex environmental samples.

Carbapenems are a class of β-lactam antibiotics with a broad antimicrobial activity spectrum. Owing to their sturdy structures resistant to most β-lactamases, they have been regarded as one of the last-resort antibiotics for combating multidrugresistant bacterial infections. However, the emergence of carbapenem resistance increases predominantly in nosocomial pathogens. To prevent spread of carbapenem resistance in early stages, it is imperative to develop rapid diagnostic tests that will substantially reduce the time and cost in determining carbapenem resistance. Thus, we devised a staining-based diagnostic method applicable to three different Gram-negative pathogens of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, all with the high potential to develop carbapenem resistance. Regardless of the resistance mechanisms presented by bacterial species and strains, double staining with propidium iodide (PI) and alamar blue (AB) identified resistant bacteria with an average sensitivity of 95.35%, 7 h after imipenem treatments in 343 clinical isolates. Among the three species tested, A. baumannii showed the highest diagnostic sensitivity of 98.46%. The PI and ABmediated staining method could be a promising diagnostic method with high-throughput efficacy and low cost.

The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 μM, 40 μM, 50 μM, 60 μM and 80 μM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 μM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 10 3 or 5.13 × 10 4 CFU/g for VBNC E. coli O157:H7 and 1.05 × 10 4 or 1.05 × 10 5 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce., (Copyright © 2020 American Society for Microbiology.)

As a novel nonthermal technology, nonthermal plasma (NTP) has attracted a lot of attention. However, it could induce microorganisms into a viable but nonculturable (VBNC) state, posing a potential risk to food safety and public health. In this study, the molecular mechanisms of VBNC Staphylococcus aureus induced by NTP were investigated. With the use of a propidium monoazide quantitative PCR (PMA-qPCR) technique combined with a plate count method, we confirmed that 8.1 to 24.3 kJ NTP induced S. aureus into a VBNC state at a level of 7.4 to 7.6 log 10 CFU/ml. The transcriptomic analysis was conducted and revealed that most energy-dependent physiological activities (e.g., metabolism) were arrested in VBNC S. aureus , while the oxidative stress response-related genes ( katA , dps , msrB , msrA , and trxA ) were significantly upregulated. In addition, this study showed that the ATP depletion by carbonyl cyanide m -chlorophenyl hydrazone (CCCP) pretreatment could accelerate the formation of VBNC S. aureus The NTP-generated oxidative stress triggers the staphylococcal oxidative stress response, which consumes part of cellular energy (e.g., ATP). The energy allocation is therefore changed, and the energy assigned for other energy-dependent physiological activities (cell growth and division, etc.) is reduced, subsequently forcing S. aureus into a VBNC state. Therefore, the alterations of energy allocation should be some of the major contributors to the induction of VBNC S. aureus with NTP exposure. This study provides valuable knowledge for controlling the formation of VBNC S. aureus during NTP treatment. IMPORTANCE In recent years, nonthermal plasma (NTP) technology has received a lot of attention as a promising alternative to thermal pasteurization in the food industry. However, little is known about the microbial stress response toward NTP, which could be a potential risk to food safety and impede the development of NTP. A viable but nonculturable (VBNC) state is one of the most common survival strategies employed by microorganisms against external stress. This study investigated the mechanisms of the formation of VBNC Staphylococcus aureus by NTP in a more comprehensive and systematic aspect than had been done before. Our work confirmed that the NTP-generated oxidative stress induced changes in energy allocation as a driving force for the formation of VBNC S. aureus This study could provide better knowledge for controlling the occurrence of VBNC S. aureus induced by NTP, which could lead to more rational design and ensure the development of safe foods., (Copyright © 2020 American Society for Microbiology.)

Measuring viability is an important and necessary assessment in studying microorganisms. Several methods can be applied to Leptospira spp., each with advantages and inconveniencies. Here, we describe the traditional colony-forming unit method, together with two other methods based, respectively, on the reducing capacity of live cells (Alamar Blue ® Assay) and differential staining of live and dead cells (LIVE/DEAD BacLight ® ). The Alamar Blue ® Assay uses the blue reagent resazurin, which can be reduced into the pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance or fluorescence reading. The LIVE/DEAD BacLight ® assay uses a mixture of two nucleic acid dyes (Syto9 and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The two other methods are not laborious and well-adapted to high-throughput studies, but the range of detection and linearity are limited.

Caveolae are plasma membrane organelles that are, among many other features, involved in mechanosensing and mechanoprotection. Different tools have been developed to study caveolae-dependent mechanoprotection and had to be adapted to the tissue or cells studied, as these structures are found in almost every type of cells. This chapter focuses on a protocol combining the use of live-cell imaging, micropatterning, hypo-osmotic shock as a mechanical stress, and dyes such as calcein-AM and propidium iodide. We used this protocol for the in vitro study of the effect of mechanical stress on membrane integrity in human muscle cells from patients bearing caveolin-3 mutations.

A series of new 4-N-phenylaminoquinoline derivatives were designed, synthesized, and their anticholinesterase activities, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, metal-chelating ability were tested. Among them, compounds 11j, 11k and 11l had comparable inhibition activities to reference drug galantamine both in AChE and in BChE. Especially, compound 11j revealed the most potent inhibition to eeAChE and eqBChE with IC 50 values of 1.20 μM and 18.52 μM, respectively. Furthermore, both kinetic analysis of AChE inhibition and molecular docking study indicated that compound 11j was mixed-type inhibitor, binding simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE, and propidium iodide displacement assay showed significant displacement of propidium iodide with compound 11k (25.80%) from PAS of eeAChE. More importantly, compound 11l displayed excellent DPPH radical scavenging activity (84% at 1 mg/mL), and its EC 50 value was 0.328 μM. In addition, compounds 11a, 11j, 11k and 11l exhibited obvious biometal chelating abilities toward Al 3+ , Fe 2+ , Cu 2+ and Zn 2+ ions. Taken together, 4-N-phenylaminoquinoline derivatives targeting multiple pathogenetic factors deserve further investigation for treatment of AD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: The purpose of this experiment was to study the bacterial diversity and predominance of spoilage bacteria in chicken skin at different thermal treatment temperatures (60, 70, 80, 90, 100, 110, 120°C)., Method and Results: Bacteria in chicken skin was collected, then propidium monoazide treatment to remove the DNA of dead cell, total DNA was extracted by Tiandz Bacterial DNA Kit, and investigated by high-throughput sequencing of the v3/v4 regions of the 16S rDNA gene. A total of 796 008 high-quality bacterial sequences were obtained for assessing the microbial diversity of chicken skin from seven thermal treatment group and control group. The results showed that the bacterial diversity in chicken skin at 90°C was lowest. And Acinetobacter (25·88%), Clostridium (20·70%), Bacteroides (13·93%) and Myroides (13·13%) were the main flora at 25°C; The Clostridium was dominant genus of the samples heat-treated by 60, 70, 80 and 90°C, the proportion of this genus were up to 64·86, 77·42, 52·22 and 87·30% respectively. The Bacillus was the main flora of the samples heat-treated by 100, 110 and 120°C, and the relative percentages were 39·44, 79·61 and 45·96% respectively. In addition, high-temperature-resistant Serratia was found in chicken skin., Conclusions: The study revealed that the relationship between thermal treatment temperature and bacterial diversity and dominant spoilage bacteria in chicken skin, which had a strong guiding significance for the control and prediction of micro-organisms in foods., Significance and Impact of the Study: The results of this paper could provide a theoretical basis for meat products containing chicken skin, including the safe use of chicken skin, determination of sterilization process parameters and selection of preservatives for compounding, which has strong practicality in China., (© 2019 The Society for Applied Microbiology.)

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log 10 viable cells ml -1 ), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria. IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens., (Copyright © 2019 American Society for Microbiology.)

Emetic Bacillus cereus is one of the causative agents of foodborne diseases which can cause vomiting-type food poisoning after ingestion of contaminated food. To minimize B. cereus food poisoning, propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) called PMA-qPCR was applied for detecting viable emetic B. cereus in milk. The cereulide synthetase gene of emetic B. cereus (cesB) was chosen for the primer, and PMA treatment was optimized at 3 μg/mL to inhibit the PCR amplification of DNA from dead cells. Under optimized assay parameters, the limit of detection (LOD) using this method were 10 2  CFU/mL in both pure culture and in spiked milk matrix. The cycle threshold (Ct) values obtained for this assay was not significantly affected by the presence of non-target bacteria such as E. coli O157:H7 which indicated the high selectivity of the assay for emetic B. cereus. The PMA-qPCR assay used in this study has the potential for sensitive detection of viable emetic B. cereus in milk., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Herein we report a simple fluorescence microscopy methodology that, jointly with four photosensitizers (PSs) and a cell viability marker, allows monitoring of phenotypic bacterial resistance to photodynamic inactivation (PDI) treatments. The PSs, composed of BODIPY dyes, were selected according to their ability to interact with the cell wall and the photoinactivating mechanism involved (type I or type II). In a first approach, the phenotypic heterogeneity allowing bacteria to persist during PDI treatment was evaluated in methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli as Gram-positive and Gram-negative models, respectively. By means of propidium iodide (PI), we monitored with spatiotemporal resolution cell viability at the single bacterium level. All the PSs were effective at inactivating pathogens; however, the cationic nonhalogenated PS (compound 1 ) surpassed the others and was capable of photoinactivating E. coli even under optimal growth conditions. Compound 1 was further tested on two other Gram-negative strains, Pseudomonas aeruginosa and Klebsiella pneumonia e , with outstanding results. All bacterial strains used here are well-known ESKAPE pathogens, which are the leading cause of nosocomial infections worldwide. Thorough data analysis of individual cell survival times revealed clear phenotypic variation expressed in the cell wall that affected PI permeation and thus its intercalation with DNA. For the same bacterial sample, death times may vary from seconds to hours. In addition, the PI incorporation time is also a parameter governed by the phenotypic characteristics of the microbes. Finally, we demonstrate that the results gathered for the bacteria provide direct and unique experimental evidence that supports the time-kill curve profiles.

We report herein the colorimetric identification of live cells based on a nucleic acid amplification testing (NAAT) methodology using an all-in-one origami paper microdevice integrated with DNA purification, loop-mediated isothermal amplification (LAMP), and on-site colorimetric detection. First, origami paper was partially embossed to create microchannel networks and chambers. Subsequently, hydrophobic polydimethylsiloxane prepolymer was coated onto the embossed paper to stabilize the structures on paper and provide fluid barriers. The paper microdevice was composed of splitting, purification, wicking, reaction, and dye pads folded alternatively to accomplish sensitive and specific NAAT. For the viability assay, propidium monoazide (PMA) was employed to penetrate dead cells and form covalent bonds with necrotic cell DNA; thus, amplification can be solely performed with DNA obtained from live bacterial cells. Purification functionality was implemented into the microdevice using chitosan to electrostatically capture DNA. Herein, methylene blue, which is typically used for electrochemical detection, is introduced for the first time for colorimetric detection of LAMP amplicons. This origami paper microdevice was successfully applied to determine the viability of foodborne pathogens, such as Escherichia coli O157:H7 and Salmonella spp., in which amplification was performed for 30 min followed by the execution of the colorimetric method for 10 min, thereby demonstrating tremendous potential for multiplexing and versatility for point-of-care applications. The introduced origami paper microdevice could be an attractive substitute as an instantaneous and convenient screening tool for the identification of viable pathogens in the control and monitoring of foodborne outbreaks in low-resource environments.

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO 9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO 9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions., (© 2019 The Society for Applied Microbiology.)

Quantitative Polymerase Chain Reaction (qPCR) is a molecular method commonly used to detect and quantify bacterial DNA on food but is limited by its inability to distinguish between live and dead cell DNA. To overcome this obstacle, propidium monoazide (PMA) alone or with deoxycholate (DC) was used to prevent dead cell detection in qPCR. qPCR methods were used to detect strains of Escherichia coli O157, which can cause infection in humans with an infectious dose of less than 10 cells. A 5 strain E. coli O157:H7 cocktail was inoculated onto beef steaks and treated with interventions used in meat facilities (lactic acid (5%), peroxyacetic acid (200 ppm) or hot water (80 °C for 10 s)). Treatment of PMA or PMA + DC was applied to samples followed by DNA extraction and quantification in qPCR. RNA was also quantified in addition to conventional plating. For lactic acid intervention, qPCR DNA quantification of E. coli O157:H7 yielded 6.59 ± 0.21 and 6.30 ± 0.11 log gene copy #/cm 2 for control and lactic acid samples, respectively and after treatment with PMA or PMA + DC this was further reduced to 6.31 ± 0.21 and 5.58 ± 0.38, respectively. This trend was also observed for peroxyacetic acid and hot water interventions. In comparison, RNA quantification yielded 7.65 ± 0.13 and 7.02 ± 0.38 log reverse transcript/cm 2 for rRNA control and lactic acid samples, respectively, and for plating (LB), 7.51 ± 0.06 and 6.86 ± 0.32 log CFU/cm 2 , respectively. Our research determined that treatment of PMA + DC in conjunction with qPCR prevented dead cell DNA detection. However, it also killed cells injured from intervention that may have otherwise recovered. RNA quantification was more laborious and results had higher variability. Overall, quantification with conventional plating proved to be the most robust and reliable method for live EHEC detection on beef., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids (eNA). We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at eNA being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)-qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99 °C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts' dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45 °C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA-qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA-qPCR is able to assess the impact of heating on T. gondii oocysts' viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.