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 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.

A flow cytometry method for safe detection of bacterial viability.

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.

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.

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.

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.

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.

Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant.

BACKGROUND: Gut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs. METHODS: LGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle. RESULTS: We demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action. CONCLUSION: Overall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.

Prediction of Spheroid Cell Death Using Fluorescence Staining and Convolutional Neural Networks.

Three-dimensional (3D) cell culture is emerging for drug design and drug screening. Skin toxicity is one of the most important assays for determining the toxicity of a compound before being used in skin application. Much work has been done to find an alternative assay without animal experiments. 3D cell culture is one of the methods that provides clinically relevant models with superior clinical translation compared to that of 2D cell culture. In this study, we developed a spheroid toxicity assay using keratinocyte HaCaT cells with propidium iodide and calcein AM. We also applied the transfer learning-containing convolutional neural network (CNN) to further determine spheroid cell death with fluorescence labeling. Our result shows that the morphologies of the spheroid are the key features in determining the apoptosis cell death of the HaCaT spheroid. Our CNN model provided good statistical measurement in terms of accuracy, precision, and recall in both validation and external test data sets. One can predict keratinocyte spheroid cell death if that spheroid image contains the fluorescence signals from propidium iodide and calcein AM. The CNN model can be accessed in the web application at https://qsarlabs.com/#spheroiddeath.

Effect of active vitamin D on proliferation, cell cycle and apoptosis in endometriotic stromal cells.

RESEARCH QUESTION: What is the effect of vitamin D3 (1,25(OH)2D3) on proliferation, cell cycle and apoptosis of endometrial stromal cells (ESC) in endometriotic patients? DESIGN: ESC isolated from 10 women with endometriosis and 10 healthy controls were treated with 1,25(OH)2D3. The proliferation of control endometrial stromal cells (CESC), eutopic endometrial stromal cells (EuESC) and ectopic endometrial stromal cells (EESC) was analysed 72 h after the treatment using methyl thiazolyl tetrazolium assay. Propidium iodide staining and flow cytometry were used to determine the cell cycle distribution in ESC. Annexin V/propidium iodide double staining was used to evaluate apoptosis in ESC. RESULTS: In the presence of oestrogen, 1,25(OH)2D3 treatment inhibited the proliferation of ESC from all three origins (P = 0.009 for CESC, P = 0.005 for EuESC and P < 0.001 for EESC). The percentage of S phase cells in EESC was higher than in EuESC and CESC (P = 0.002 and P = 0.001, respectively). The percentage of S phase cells in EuESC was higher than in CESC (P = 0.005). The percentage of G1 phase cells in EESC was lower than that of EuESC and CESC (P = 0.003 and P = 0.002, respectively) and the percentage of G1 phase cells in EuESC was lower than that of CESC (P = 0.007). Moreover, 1,25(OH)2D3 inhibited cell cycle regardless of cell type (P = 0.002 in EESC, P = 0.001 in EuESC and P = 0.014 in CESC), but in the absence of oestrogen, inhibited cell cycle only in EuESC (P = 0.012). CONCLUSIONS: Although 1,25(OH)2D3 increased apoptotic and necrotic cells and decreased live cells in the EuESC and EESC, it did not affect apoptosis in CESC and only increased necrotic cells. These findings indicate that 1,25(OH)2D3 potentially has a growth-inhibiting and pro-apoptotic effect on ESC from endometriotic patients.

Hsa_circ_0001550 impairs decidualization by regulating the proliferation and apoptosis of endometrial stromal cells.

RESEARCH QUESTION: What is the molecular function of hsa_circ_0001550 in decidualization? DESIGN: Human endometrial stromal cells (HESC) were isolated from the endometrium tissues to build an in-vitro decidualization model. Different concentrations of medroxyprogesterone acetate (MPA) were used to observe whether the expression level of hsa_circ_0001550 was related to progesterone. Biological characteristics and distribution of hsa_circ_0001550 were determined by RNase R, actinomycin D (Act D) assay and cytoplasmic/nuclear fraction assay. Then the overexpression of hsa_circ_0001550 was achieved by adenovirus vector. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assays. The cell cycle was assessed by flow cytometry analyses. Cell apoptosis was determined by annexin-V/propidium iodide double staining experiment and western blotting. RESULTS: The expression of hsa_circ_0001550 was decreased in decidua and decidualized HESC (P < 0.001, P = 0.014). Hsa_circ_0001550 is a covalently closed RNA molecule that was verified by RNase R assay and Act D assay (P = 0.012). Nuclear and cytoplasmic separation experiments confirmed that hsa_circ_0001550 was mainly distributed in the cytoplasm. Overexpression of hsa_circ_0001550 inhibited decidualization of HESC (P < 0.0001). Furthermore, overexpression of hsa_circ_0001550 inhibited proliferation by decreasing the number of S phase cells (P = 0.033). Annexin-V/propidium iodide double staining experiment and western blotting revealed that overexpression of hsa_circ_0001550 promoted HESC apoptosis (P < 0.001, P = 0.0139). CONCLUSIONS: Hsa_circ_0001550 impairs decidualization of HESC. Progesterone decreases the expression of hsa_circ_0001550. The results may provide new insights into the cause of decidualization.

Cryopreservation of human cerebral microvascular endothelial cells with glycerol.

The cryopreservation of human cerebral microvascular endothelial cells (hCMEC) has facilitated their commercial availability for research studying the blood-brain barrier. The currently employed cryopreservation protocol uses 10% dimethyl sulfoxide (Me2SO) in cell medium, or 5% Me2SO in 95% fetal bovine serum (FBS) as cryoprotective agents (CPAs). However, Me2SO is toxic to cells and FBS is animal-derived and not chemically defined, so reducing the concentrations of these components is desirable. Recently, we showed that cryopreserving hCMEC in cell medium with 5% Me2SO and 6% hydroxyethyl starch (HES) results in over 90% post-thaw cell viability. This previous work was performed using an interrupted slow cooling (graded freezing) approach followed by SYTO13/GelRed staining to assay for membrane integrity. In this paper, we repeated graded freezing of hCMEC in cell medium containing 5% Me2SO and 6% HES, but this time using Calcein AM/propidium iodide staining to ensure that the stain is an equivalent alternative to SYTO13/GelRed for assessment of cell viability, and that results are comparable to those previously published. Next, using graded freezing experiments and Calcein AM/propidium iodide staining, we examined the effectiveness of non-toxic glycerol as a CPA at different concentrations, loading times, and cooling rates. The cryobiological response of hCMEC was used to develop a protocol that optimizes both the permeating and non-permeating capabilities of glycerol. HCMEC in cell medium loaded with 10% glycerol for 1 h at room temperature, ice nucleated at -5 °C and held for 3 min, and then cooled at -1 °C/min to -30 °C before plunging into liquid nitrogen had post-thaw viability of 87.7% ± 1.8%. Matrigel tube formation assay and immunocytochemical staining of junction protein ZO-1 were carried out on post-thaw hCMEC to ensure that the cryopreserved cells were viable and functional, in addition to being membrane-intact.

A comparative analysis of quantitative detection methods for viable food-borne pathogens using RT-qPCR and PMA-qPCR.

The accurate quantification of viable pathogens in food is crucial for ensuring food safety. This study mainly aimed to investigate the quantification of viable pathogens using PMA-qPCR and RT-qPCR, taking into account bacterial species, food matrices, and inactivation methods. The detection limit of PMA-qPCR for Salmonella serovars in simple matrices, such as culture broth, lake, or tap water, was found to be 102 cells per ml. Regarding the detection of Staphylococcus aureus and Escherichia coli in culture broth, as well as Salmonella in more complex matrices, such as juices and lab-made broth, both methods exhibited a detection limit of 103 cells per ml. Besides that, in adverse situations, there was a risk of overestimating the number of viable pathogens using PMA-qPCR. In addition, a conspicuous discrepancy between the results of PMA-qPCR/RT-qPCR and those of the plate counting assay was observed when Salmonella was exposed to isopropanol, H2O2, NaClO, sonication, or thermosonication. This suggests that it may survive in a viable but non-culturable state and poses a challenge for accurate quantification of viable cells using plate counting assay. Therefore, the results obtained by RT-qPCR were more objective compared to PMA-qPCR due to potential influences from bacteria species, surrounding media, and inactivation methods.

A Pilot Study on the Uptake of Propidium Iodide and YO-PRO-1 Iodide through the Pannexin Channels in Wallachian Frozen-Thawed Ram Spermatozoa.

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p < .05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p < .05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p < .05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

Development of a Propidium Monoazide-Based Viability Quantitative PCR Assay for Red Sea Bream Iridovirus Detection.

Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 µM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.

Effects and Mechanisms Activated by Treatment with Cationic, Anionic and Zwitterionic Liposomes on an In Vitro Model of Porcine Pre-Pubertal Sertoli Cells.

Liposomes have been successfully used as drug-delivery vehicles, but there are no clinical studies on improved fertility and the few reported experimental studies have been performed in animal models far from humans. The aim of this paper was to study the effects of treatment with cationic, anionic and zwitterionic liposomes on our superior mammalian model of porcine prepubertal Sertoli cells (SCs) to find a carrier of in vitro test drugs for SCs. Porcine pre-pubertal SCs cultures were incubated with different liposomes. Viability, apoptosis/necrosis status (Annexin-V/Propidium iodide assay), immunolocalisation of ß-actin, vimentin, the phosphorylated form of AMP-activated protein Kinase (AMPK)α and cell ultrastructure (Transmission Electron Microscopy, TEM) were analysed. Zwitterionic liposomes did not determine changes in the cell cytoplasm. The incubation with anionic and cationic liposomes modified the distribution of actin and vimentin filaments and increased the levels of the phosphorylated form of AMPKα. The Annexin/Propidium Iodide assay suggested an increase in apoptosis. TEM analysis highlighted a cytoplasmic vacuolisation. In conclusion, these preliminary data indicated that zwitterionic liposomes were the best carrier to use in an in vitro study of SCs to understand the effects of molecules or drugs that could have a clinical application in the treatment of certain forms of male infertility.

Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selectively suppress the DNA amplification of dead cells. The results showed the optimum concentration of PMA is 5.0 µg/mL. The detection limit of this assay by mPCR was 103 CFU/mL in the culture broth, and by PMA-mPCR was 104 CFU/mL both in pure culture and a food matrix (milk and ground beef). In addition, the detection of mixed viable and dead cells was also explored in this study. The detection sensitivity ratio of viable and dead counts was less than 1:10. Therefore, the PMA-mPCR assay proposed here might provide an efficient detection tool for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella and also have great potential for the detection and concentration assessment of VBNC cells.

Establishment of methods to analyze the structural and functional integrity of the quail (Coturnix coturnix japonica) sperm plasma membrane.

1. The objectives of this study were to establish the use of the fluorophores Hoechst 33342 and propidium iodide for the evaluation of sperm plasma membrane integrity and to identify an adequate hypoosmotic solution for the evaluation of sperm membrane functionality in quails.2. Sperm samples were collected from the vas deferens of nine quails. After initial evaluation, the samples were subjected to a flash-frozen assay. Three treatments with the following proportions of fresh sperm and sperm subjected to flash freezing were prepared as follows: 100:0 (T100), 50:50 (T50), and 0:100 (T0). The hypoosmotic swelling test used distilled water (0 mOsm/l) and fructose solutions (50, 100, and 200 mOsm/l).3. Immediately after recovery, the samples showed 75.6 ± 5.0% motility with vigour of 3.7 ± 0.3 and 96.1 ± 0.5% of the sperm appeared normal. The membrane integrity test showed 62.2 ± 5.2% intact sperm at T100, 29.0 ± 4.1% at T50 and 0.1 ± 0.1% at T0. Moreover, a greater number of reactive sperm (74.7 ± 6.7%) were observed when incubated in distilled water (0 mOsm/l) in comparison to other solutions (P < 0.05).4. The association of fluorescent probes composed of Hoechst 33342 and propidium iodide provided an efficient assessment of the integrity of the plasmatic membrane of quail spermatozoa. However, the study identified that the hypoosmotic swelling test has little predictive value regarding sperm membrane functionality in this species.

Propidium uptake and ATP release in A549 cells share similar transport mechanisms.

The lipid bilayer of eukaryotic cells' plasma membrane is almost impermeable to small ions and large polar molecules, but its miniscule basal permeability in intact cells is poorly characterized. This report describes the intrinsic membrane permeability of A549 cells toward the charged molecules propidium (Pr2+) and ATP4-. Under isotonic conditions, we detected with quantitative fluorescence microscopy, a continuous low-rate uptake of Pr (∼150 × 10-21 moles (zmol)/h/cell, [Pr]o = 150 µM, 32°C). It was stimulated transiently but strongly by 66% hypotonic cell swelling reaching an influx amplitude of ∼1500 (zmol/h)/cell. The progressive Pr uptake with increasing [Pr]o (30, 150, and 750 µM) suggested a permeation mechanism by simple diffusion. We quantified separately ATP release with custom wide-field-of-view chemiluminescence imaging. The strong proportionality between ATP efflux and Pr2+ influx during hypotonic challenge, and the absence of stimulation of transmembrane transport following 300% hypertonic shock, indicated that ATP and Pr travel the same conductive pathway. The fluorescence images revealed a homogeneously distributed intracellular uptake of Pr not consistent with high-conductance channels expressed at low density on the plasma membrane. We hypothesized that the pathway consists of transiently formed water pores evenly spread across the plasma membrane. The abolition of cell swelling-induced Pr uptake with 500 µM gadolinium, a known modulator of membrane fluidity, supported the involvement of water pores whose formation depends on the membrane fluidity. Our study suggests an alternative model of a direct permeation of ATP (and other molecules) through the phospholipid bilayer, which may have important physiological implications.