Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopy Observation.

Foodborne pathogens are responsible for foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can be entered in a viable but nonculturable (VBNC) state. VBNC cells are characterized by an active metabolism and a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples and thus may pose a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method combining propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain.

Fast and sensitive detection of viable Escherichia coli O157:H7 using a microwell-confined and propidium monoazide-assisted digital CRISPR microfluidic platform.

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 × 101 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.

A reliable qPCR technique for detecting viable Xanthomonas arboricola pv. pruni cells.

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.

Growth Phase Contribution in Dictating Drug Transport and Subcellular Accumulation inside Escherichia coli.

Depending upon nutrient availability, bacteria transit to multiple growth phases. The transition from the active to nongrowing phase results in reduced drug efficacy and, in some cases, even multidrug resistance. However, due to multiple alterations in the cell envelope, probing the drug permeation kinetics during growth phases becomes perplexing, especially across the Gram-negative bacteria's complex dual membrane envelope. To advance the understanding of drug permeation during the life cycle of Gram-negative bacteria, we sought to address two underlying objectives: (a) how changes are occurring inside the bacterial envelope during growth and (b) how the drug permeation and accumulation vary across both the membranes and in subcellular compartments during growth. Both objectives are met with the help of nonlinear optical technique second-harmonic generation spectroscopy (SHG). Specifically, using SHG, we probed the transport kinetics and accumulation of a quaternary ammonium compound (QAC), malachite green, inside Escherichia coli in various growth phases. Further insight about another QAC molecule, propidium iodide, is accomplished using fluorescence microscopy. Results indicate that actively growing cells have faster drug transport and higher cytoplasmic accumulation than slow- or nongrowing cells. In this regard, the rpoS gene plays a crucial role in limiting drug transport across the saturation phase cultures. Moreover, within a particular growth phase, membrane permeability undergoes gradual changes much before the subsequent growth phase commences. These outcomes signify the importance of reporting the growth phase and rate in drug efficacy studies.

Determination of airborne influenza virus and coronavirus infectivity using capsid integrity polymerase chain reaction.

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 (PtCl4) were used to investigate the effects of reagent concentration. Feasibility tests revealed that PtCl4 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.

Rapid determination of antibiotic susceptibility of clinical isolates of Escherichia coli by SYBR green I/Propidium iodide assay.

Infections caused by pathogenic Escherichia coli are a serious threat to human health, while conventional antibiotic susceptibility tests (AST) have a long turn-around time, and rapid antibiotic susceptibility methods are urgently needed to save lives in the clinic, reduce antibiotic misuse and prevent emergence of antibiotic-resistant bacteria. We optimized and validated the feasibility of a novel rapid AST based on SYBR Green I and Propidium Iodide (SGPI-AST) for E. coli drug susceptibility test. A total of 112 clinical isolates of E. coli were collected and four antibiotics (ceftriaxone, cefoxitin, imipenem, meropenem) were selected for testing. Bacterial survival rate of E. coli was remarkably linearly correlated with S value at different OD600 values. After optimizing the antibiotic concentrations, the sensitivity and specificity of SGPI-AST reached 100%/100%, 97.8%/100%, 100%/100% and 98.4%/99% for ceftriaxone, cefoxitin, imipenem and meropenem, respectively, and the corresponding concordances of the SGPI-AST with conventional AST were 1.000, 0.980, 1.000 and 0.979, respectively. The SGPI-AST can rapidly and accurately determine the susceptibility of E. coli clinical isolates to multiple antibiotics in 60 min, and has the potential to be applied to guide the precise selection of antibiotics for clinical management of infections caused by pathogenic E. coli.

Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness. IMPORTANCE: This study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

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.

Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR.

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.

Comparison of plate counting with flow cytometry, using four different fluorescent dye techniques, for the enumeration of Bacillus cereus in milk.

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 DiOC2 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 DiOC2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC2 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 DiOC2 dye, to determine the viable number of cells, it was found that the DiOC2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis.

PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

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.

Development of a Long-Amplicon Propidium Monoazide-Quantitative PCR Assay for Detection of Viable Xanthomonas arboricola pv. pruni Cells in Peach Trees.

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 103 CFU/ml with the presence of up to 107 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 (R2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees.

The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity.

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.

Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR.

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

The investigation of cytotoxic and apoptotic activity of Cl-amidine on the human U-87 MG glioma cell line.

BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors. METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry. RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma. CONCLUSION: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.

Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Assessing chronological aging in Saccharomyces cerevisiae.

Chronological age represents the time that passes between birth and a given date. To understand the complex network of factors contributing to chronological lifespan, a variety of model organisms have been implemented. One of the best studied organisms is the yeast Saccharomyces cerevisiae, which has greatly contributed toward identifying conserved biological mechanisms that act on longevity. Here, we discuss high- und low-throughput protocols to monitor and characterize chronological lifespan and chronological aging-associated cell death in S. cerevisiae. Included are propidium iodide staining with the possibility to quantitatively assess aging-associated cell death via flow cytometry or qualitative assessments via microscopy, cell viability assessment through plating and cell counting and cell death characterization via propidium iodide/AnnexinV staining and subsequent flow cytometric analysis or microscopy. Importantly, all of these methods combined give a clear picture of the chronological lifespan under different conditions or genetic backgrounds and represent a starting point for pharmacological or genetic interventions.

On-site filtration of large sample volumes improves the detection of opportunistic pathogens in drinking water distribution systems.

In this study, we compared conventional vacuum filtration of small volumes through disc membranes (effective sample volumes for potable water: 0.3-1.0 L) with filtration of high volumes using ultrafiltration (UF) modules (effective sample volumes for potable water: 10.6-84.5 L) for collecting bacterial biomass from raw, finished, and tap water at seven drinking water systems. Total bacteria, Legionella spp., Legionella pneumophila, Mycobacterium spp., and Mycobacterium avium complex in these samples were enumerated using both conventional quantitative PCR (qPCR) and viability qPCR (using propidium monoazide). In addition, PCR-amplified gene fragments were sequenced for microbial community analysis. The frequency of detection (FOD) of Legionella spp. in finished and tap water samples was much greater using UF modules (83% and 77%, respectively) than disc filters (24% and 33%, respectively). The FODs for Mycobacterium spp. in raw, finished, and tap water samples were also consistently greater using UF modules than disc filters. Furthermore, the number of observed operational taxonomic units and diversity index values for finished and tap water samples were often substantially greater when using UF modules as compared to disc filters. Conventional and viability qPCR yielded similar results, suggesting that membrane-compromised cells represented a minor fraction of total bacterial biomass. In conclusion, our research demonstrates that large-volume filtration using UF modules improved the detection of opportunistic pathogens at the low concentrations typically found in public drinking water systems and that the majority of bacteria in these systems appear to be viable in spite of disinfection with free chlorine and/or chloramine.IMPORTANCEOpportunistic pathogens, such as Legionella pneumophila, are a growing public health concern. In this study, we compared sample collection and enumeration methods on raw, finished, and tap water at seven water systems throughout the State of Minnesota, USA. The results showed that on-site filtration of large water volumes (i.e., 500-1,000 L) using ultrafiltration membrane modules improved the frequency of detection of relatively rare organisms, including opportunistic pathogens, compared to the common approach of filtering about 1 L using disc membranes. Furthermore, results from viability quantitative PCR (qPCR) with propidium monoazide were similar to conventional qPCR, suggesting that membrane-compromised cells represent an insignificant fraction of microorganisms. Results from these ultrafiltration membrane modules should lead to a better understanding of the microbial ecology of drinking water distribution systems and their potential to inoculate premise plumbing systems with opportunistic pathogens where conditions are more favorable for their growth.

Inactivation of Human Norovirus GII.4's Infectivity in Fresh Oysters (Crassostrea gigas) through Thermal Treatment in Association with Propidium Monoazide.

The current study investigated the effects of heat treatment (85 °C or 100 °C for 5-20 min) on human norovirus (HuNoV) GII.4's capsid stability in fresh oysters. In addition, propidium monoazide (PMA) was used in viral samples to distinguish infectious viruses and evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Further, we explored the effect of the heat treatment on oyster quality (Hunter color and hardness). The titer of HuNoV for oysters significantly (p < 0.05) decreased to 0.39-1.32 and 0.93-2.27 log10 copy number/µL in the non-PMA and PMA-treated groups, respectively, after heat treatment. HuNoV in oysters not treated with PMA showed a decrease of <1.5 - log10, whereas in PMA-treated oysters, a decrease of >1 - log10 was observed after treatment at 85 °C for 10 min. Treatments for both 15 min and 20 min at 100 °C showed a >99% log10 reduction using PMA/RT-qPCR. In the Hunter color, an increase in heat temperature and duration was associated with a significant decrease in 'L' (brightness+, darkness-) and an increase in 'a' (redness+, greenness-) and 'b' (yellowness+, blueness-) (p < 0.05). Our findings confirmed that the hardness of oyster meat significantly increased with increasing temperature and time (p < 0.05). This study demonstrated that PMA/RT-qPCR was effective in distinguishing HuNoV viability in heat-treated oysters. The optimal heat treatment for oysters was 10 min at 85 °C and 5 min at 100 °C.

Rapid and Accurate Quantification of Viable Lactobacillus Cells in Infant Formula by Flow Cytometry Combined with Propidium Monoazide and Signal-Enhanced Fluorescence In Situ Hybridization.

Lactobacillus is an important member of the probiotic bacterial family for regulating human intestinal microflora and preserving its normalcy, and it has been widely used in infant formula. An appropriate and feasible method to quantify viable Lactobacilli cells is urgently required to evaluate the quality of probiotic-fortified infant formula. This study presents a rapid and accurate method to count viable Lactobacilli cells in infant formula using flow cytometry (FCM). First, Lactobacillus cells were specifically and rapidly stained by oligonucleotide probes based on a signal-enhanced fluorescence in situ hybridization (SEFISH) technique. A DNA-binding fluorescent probe, propidium monoazide (PMA), was then used to accurately recognize viable Lactobacillus cells. The entire process of this newly developed PMA-SEFISH-FCM method was accomplished within 2.5 h, which included pretreatment, dual staining, and FCM analysis; thus, this method showed considerably shorter time-to-results than other rapid methods. This method also demonstrated a good linear correlation (R2 = 0.9994) with the traditional plate-based method with a bacterial recovery rate of 91.24%. To the best of our knowledge, the present study is the first report of FCM combined with PMA and FISH for the specific detection of viable bacterial cells.