N-3-Methylbutyl-benzisoselenazol-3(2H)-one Exerts Antifungal Activity In Vitro and in a Mouse Model of Vulvovaginal Candidiasis.

In the present work, we evaluated the antifungal activities of two novel ebselen analogs, N-allyl-benzisoselenazol-3(2H)-one (N-allyl-bs) and N-3-methylbutylbenzisoselenazol-3(2H)-one (N-3mb-bs). Colorimetric and turbidity assays were performed to determine the minimum inhibitory concentration (MIC) of these compounds in S1 (fluconazole-sensitive) and S2 (fluconazole-resistant) strains of C. albicans. N-3mb-bs was more active than the N-allyl-bs compound. It is noteworthy that the concentration of N-3mb-bs observed to inhibit fungal growth by 50% (18.2 µM) was similar to the concentration observed to inhibit the activity of the yeast plasma membrane H+-ATPase (Pma1p) by 50% (19.6 µM). We next implemented a mouse model of vulvovaginal candidiasis (VVC) using the S1 strain and examined the mouse and yeast proteins present in the vaginal lavage fluid using proteomics. The yeast proteins detected were predominately glycolytic enzymes or virulence factors associated with C. albicans while the mouse proteins present in the lavage fluid included eosinophil peroxidase, desmocollin-1, and gasdermin-A. We then utilized the N-3mb-bs compound (12.5 mg/kg) in the mouse VVC model and observed that it significantly reduced the vaginal fungal burden, histopathological changes in vagina tissue, and expression of myeloperoxidase (MPO). All in all, the present work has identified a potentially promising drug candidate for VVC treatment.

The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum.

The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum, we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2), and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae, Ptk2 knockout strains were more sensitive to terbinafine compared with the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. Pma1 is activated through phosphorylation by Ptk2 in S. cerevisiae. Overexpression of T. rubrum Pma1 (TrPma1) in T. rubrum Ptk2 knockout strain (ΔTrPtk2) suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole, an inhibitor of plasma membrane proton pump Pma1, increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine-resistant dermatophytes.

Kilogram-Scale Synthesis of Extremely Small Gadolinium Oxide Nanoparticles as a T1-Weighted Contrast Agent for Magnetic Resonance Imaging.

Magnetic resonance imaging contrast agents are frequently used in clinics to enhance the contrast between diseased and normal tissues. The previously reported poly(acrylic acid) stabilized exceedingly small gadolinium oxide nanoparticles (ES-GdON-PAA) overcame the problems of commercial Gd chelates, but limitations still exist, i.e., high r2/r1 ratio, long blood circulation half-life, and no data for large scale synthesis and formulation optimization. In this study, polymaleic acid (PMA) is found to be an ideal stabilizer to synthesize ES-GdONs. Compared with ES-GdON-PAA, the PMA-stabilized ES-GdON (ES-GdON-PMA) has a lower r2/r1 ratio (2.05, 7.0 T) and a lower blood circulation half-life (37.51 min). The optimized ES-GdON-PMA-9 has an exceedingly small particle size (2.1 nm), excellent water dispersibility, and stability. A facile, efficient, and environmental friendly synthetic method is developed for large-scale synthesis of the ES-GdONs-PMA. The weight of the optimized freeze-dried ES-GdON-PMA-26 synthesized in a 20 L of reactor reaches the kilogram level. The formulation optimization is also finished, and the concentrated ES-GdON-PMA-26 formulation (CGd = 100 mm) after high-pressure steam sterilization possesses eligible physicochemical properties (i.e., pH value, osmolality, viscosity, and density) for investigational new drug application.

Regulatory Aspects of AI-ML.

In the realm of healthcare, numerous generative and non-generative artificial intelligence and machine learning (AI-ML) tools have been developed and deployed. Simultaneously, manufacturers of medical devices are leveraging AI-ML. However, the adoption of AI in healthcare raises several concerns, including safety, security, ethical biases, accountability, trust, economic impact, and environmental effects. Effective regulation can mitigate some of these risks, promote fairness, establish standards, and advocate for more sustainable AI practices. Regulating AI tools not only ensures their safe and effective adoption but also fosters public trust. It is important that regulations remain flexible to accommodate rapid advances in this field to support innovation and also not to add additional burden to some of our preexisting and well-established frameworks. This article covers regional and global regulatory aspects of AI-ML including data privacy, Software as a Medical device (SaMD), agency approval and clearance pathways, reimbursement, and laboratory developed tests (LDTs).

Women alone modern contraceptive use decision making and its correlates, evidence from PMA CS 2021 survey.

BACKGROUND: Women alone contraceptive decisions making has become one of the top burring public health agenda. Despite Contraceptive method options are available and accessible, contraceptive prevalence rate (CPR) in Ethiopia is not far beyond 41%. Evidences showed that the freedom of women to choose the contraceptive method they desired to use is one of the potential determinants for the sluggish pace of increase in contraceptive usage. In this era of sustainable development, determining the level of women own contraceptive use decision making and identifying its correlates is very critical for the ministries and relevant partners' effort in tracking the achievement of Sustainable Development Goal (SDG) 5.2 by providing actionable evidence through informed decision-making with the aim of improving contraceptive uptake; reducing maternal mortality and improve newborn health. METHODS: Nationally representative cross-sectional data from Performance Monitoring for Action (PMA) 2021 was used in this study. The sample was restricted among2446 married women who have been using or most recently used modern contraceptive method. Cell sample size adequacy was checked using a chi-square test. Frequency was computed to characterize the study participants. Multilevel binary logistics regression was used to identify factors associated with women own contraceptive use decision making. The findings were presented in a form of frequencies, percentage and as an odds ratio using 95% confidence interval. A p-value of 0.05 was used to declare significance. RESULTS: This study revealed that higher than one in two women (59.49%; 95% CI: 57.7-61.38%) decide their contraceptive use by themselves. What is more interesting is that 1 in 16 women (6.06%) reported that they did not participated in their contraceptive use decision-making.-. Women aged 20 to 24 years; (AOR: 2.51 (1.04, 4.45)), women who stayed10 and above years in marriage; (AOR: 1.73 (1.08, 2.77)), whose husband and/or partner age is 41 and above years; (AOR: 2.14 (1.06, 4.31)) and those who obtained contraceptive method they desired; (AOR: 2.49 (1.36, 4.57)) had higher odds of deciding their current and/or recent contraceptive use by their own. On the other hand, women mixed feeling if they became pregnant at the time of the survey; (AOR: 0.6 (0.44, 0.91)), women who started using contraceptive at younger age, 19 to 24; (AOR: 0.6 (0.44, 0.81)), those who use long acting and/or permanent method; (AOR: 0.54 (0.41, 0.71)) and those married at younger age, 10 to 19 years; (AOR: 0.28 (0.09, 0.86)) had lower odds of independently deciding their current and/or most recent contraceptive use. CONCLUSION: 59% of women independently decide their contraceptive use which calls up on further improvement to enable each woman to decide by their own, with directing special focus for the 6.06% of women who reported no say in their contraceptive use decision. Activities targeting on enabling women to use the method they preferred, spacing their pregnancy, encouraging women to discuss with their husband on the time and type of contraceptive method they used, advocating and promoting marriage at least to be at the minimum age as indicate by the law and maintain the marriage duration as much as longer are hoped to improve women alone contraceptive use decision making to the fullest.

Emotional Fertility Intention and its correlates in Ethiopia among married contraceptive user women: using linked community and health facility data from performance monitoring for action; a generalized ordered logistics regression modeling.

BACKGROUND: Emotional fertility intention and couples communication are key during pregnancy and childbirth with simultaneous minimization of reproductive coercion. Intention to conceive is an integral part of the reproductive health (RH) right and can be considered as decision making on fertility, family wellbeing and the country's population demographic dividend and composition. However, in low and middle income countries including Ethiopia where males dominance is culturally constructed and socially accepted, males took the lead in every decision making process. In the aforementioned context, women are less likely for their voices to be heard, hence, this study aimed at determining the level of womens´ emotional fertility readiness and its correlates. The finding provided actionable evidence for the ministry and developmental partners working on reproductive and womens´ health so as to be used as an action point to empower women in terms of their reproductive health right to have control over their fertility. METHODS: Linked community and facility data with nationally representation from Performance Monitoring for Action (PMA Ethiopia) 2020 Survey Ethiopia except Tigray Region were used for this study. A total of 2,069 current and/or recent contraceptive user women of child bearing age who are currently married/living together as a partner were included in this analysis. Frequency was computed to describe the study participant's characteristics. Generalized Ordered logistics regression modeling was employed to identify correlates of the hierarchical variation in women fertility intention if they became pregnant. Results were presented in the form of percentages and odds ratio with 95% Confidence Intervals. Candidate variables were selected using p-value of 0.25. Statistical significance was declared at p-value of 0.05. RESULTS: The proportion of womens´ emotional fertility intention of feeling unhappiness was 48.73% (95%CI: 46.21%, 51.23%). On the contrary, 22.88%, 11.36% and 17.03% of them reported that they felt sort of happy, very happy and mixed feeling. An increase in age,10 and above years marriage duration, the type of decision maker for contraceptive use were found to increase the odds of women emotional fertility intention across the higher level categories by (AOR: 95% CI: 6.75 (3.11, 14.62) times higher among elder women aged 35 to 49 years, (AOR: 95% CI: 3.79 (1.72, 8.31) times higher for women with a 10 or more years of marriage duration; and 1.83 (1.03,3.24) times higher for women whose contraceptive use was decided by the health care provide alone. A higher birth order lowered the cumulative odds of womens´ emotional fertility intention symmetrically across the higher level categories by 86% (AOR: 95% CI: 0.14 (0.07, 0.29). Women who wanted to have additional child and whose nearest facility provided 5 or more methods had an increased odds of being in the higher level categories of women emotional fertility intention with disproportional association across the cumulative logit. Accordingly, women whose nearest health facility provided 5 or more methods had an 49% (AOR: 95%CI:1.49 (1.01, 2.19) increased likelihood of being in the mixed or happy category than being very/sort of unhappy category of the emotional fertility intention while the number of methods had no significant association with emotional fertility intention at higher cumulative logit: 1.34 (0.87,2.10). Those who wanted to have an additional child had a 3.16 (2.28, 4.36) higher odds to be in the mixed or happy category than being in unhappy category. Further, this tendency was even stronger at higher categories of emotional fertility intention: 4.83 (3.23, 7.23). CONCLUSION: Nearly one in two women reported being unhappy while 17.03% felt mixed emotion calling up on intended and spaced pregnancies by ensuring women reproductive and economic empowerment to empower women to have control over their fertility. Activities and efforts that promote intended and spaced pregnancies; and diversifying access to contraceptive methods in the nearest health facilities are likely to improve women emotional fertility intention; and activities that enable women to decide their contraceptive as well. The finding that health care provider decides on women current/recent contraceptive use calls for activities to improve quality of contraceptive use counseling to enable women to decide their contraceptive use by the themselves while the access of diversified methods in the nearby health facility create an opportunity for women to obtain the method they preferred to use and make them emotionally well. These activities are hoped to enable women to plan their fertility thereby increasing their emotional well-being. These activities and interventions need to be tailored across regions and need to be age sensitive.

Spatial distribution and determinants of Early sexual initiation in Ethiopia.

INTRODUCTION: Early sexual initiation has negative health, social, and economic consequences for both women and future generations. The trend of early sexual initiation is increasing globally, leading to higher rates of sexually transmitted diseases and unplanned pregnancies. Ethiopia has been challenged various disasters that makes women vulnerable and position them at heightened risk of early sexual initiation in the last four years. The spatial patterns and factors of early sexual initiation in the post-conflict-post pandemic settings is not well understood. Hence this research aimed at mapping Spatial Patterns and identifying determinant factors in the Post-COVID-Post-Conflict Settings. METHODS: The study was conducted on secondary data from the PMA 2021 cross-sectional survey which conducted nationally from November 2021 to January 2022 which is in the post pandemic and post-war period. Total weighted sample of 6,036 reproductive age women were included in the analysis. ArcGIS Pro and SaTScan software were used to handle spatial analysis. Multilevel logistic regression model was used to estimate the effects of independent variables on early sexual initiation at individual and community level factors. Adjusted odds ratio with the 95% confidence interval was reported to declare the strength and statistical significance of the association. RESULT: The spatial distribution of early sexual initiation was clustered in Ethiopia with a global Moran's I index value of 0.09 and Z-score 6.01 (p-value < 0.001).Significant hotspots were detected in East Gojjam zone of Amhara region, Bale, Arsi, West Hararge, East Wellega and Horo Gudru Wellega zones of Oromia region. The odds of having early sexual initiation was higher in women with primary education (AOR = 1.23, 95%CI: 1.03, 1.47), secondary or above education (AOR = 4.36, 95%CI: 3.49, 5.44), Women aged 26 to 25 (AOR = 1.91, 95%CI: 1.61, 2.26), women aged 36 to 49(AOR = 1.51, 95%CI: 1.24, 1.84). However, there was a significant lower likelihood of early sexual initiation in rural resident women (AOR = 0.53, 95%CI: 0.35, 0.81) and women living in 5 to 7 family size (AOR = 0.79, 95%CI: 0.68, 0.92), and more than 7 members (AOR = 0.63, 95%CI: 0.49, 0.81). CONCLUSIONS: The spatial distribution of early sexual initiation was clustered in Ethiopia. Interventions should be taken to eliminate the observed variation by mobilizing resources to high-risk areas. Policies and interventions targeted to this problem may also take the identified associated factors into account for better results.

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.

The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and ß-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Photocatalytic Overall Water Splitting with a Solar-to-Hydrogen Conversion Efficiency Exceeding 2% through Halide Perovskite.

Photocatalytic water splitting using semiconductors is a promising approach for converting solar energy to clean energy. However, challenges such as sluggish water oxidation kinetics and limited light absorption of photocatalyst cause low solar-to-hydrogen conversion efficiency (STH). Herein, we develop a photocatalytic overall water splitting system using I3-/I- as the shuttle redox couple to bridge the H2-producing half-reaction with the O2-producing half-reaction. The system uses the halide perovskite of benzylammonium lead iodide (PMA2PbI4, PMA = C6H5CH2NH2) loaded with MoS2 (PMA2PbI4/MoS2) as the H2 evolution photocatalyst, and the RuOx-loaded WO3 (WO3/RuOx) as the O2 evolution photocatalyst, achieving a H2/O2 production in stoichiometric ratio with an excellent STH of 2.07%. This work provides a detour route for photocatalytic water splitting with the help of I3-/I- shuttle redox couple in the halide perovskite HI splitting system and enlightens one to integrate and utilize multi catalytic strategies for solar-driven water splitting.

Identification of Aly1 and Aly2 as Modulators of Cytoplasmic pH in Saccharomyces cerevisiae.

The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell function and viability. In yeast, protons (H+) can be excreted from the cell by plasma membrane ATPase PMA1 and pumped into vacuoles by vacuolar H+-ATPase. Because PMA1 is critical to the survival of yeast cells, it is unknown whether other compensatory components are involved in pH homeostasis in the absence of PMA1. To elucidate how intracellular pH is regulated independently of PMA1, we employed a screening approach by exposing the yeast haploid deletion mutant library (ver 4.0) to the selective plant plasma membrane H+-ATPase inhibitor PS-1, which we previously reported. After repeated screenings and verification, we identified two proteins, Aly1 and Aly2, that play a role in the regulation of intracellular pH when PMA1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to PMA1 and also preliminarily reveals a role for Aly1 and Aly2 in the regulation of intracellular pH.

A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes.

From the discovery of the first membrane-interacting polymer, styrene maleic-acid (SMA), there has been a rapid development of membrane solubilising polymers. These new polymers can solubilise membranes under a wide range of conditions and produce varied sizes of nanoparticles, yet there has been a lack of broad comparison between the common polymer types and solubilising conditions. Here, we present a comparative study on the three most common commercial polymers: SMA 3:1, SMA 2:1, and DIBMA. Additionally, this work presents, for the first time, a comparative characterisation of polymethacrylate copolymer (PMA). Absorbance and dynamic light scattering measurements were used to evaluate solubilisation across key buffer conditions in a simple, adaptable assay format that looked at pH, salinity, and divalent cation concentration. Lipid-polymer nanoparticles formed from SMA variants were found to be the most susceptible to buffer effects, with nanoparticles from either zwitterionic DMPC or POPC:POPG (3:1) bilayers only forming in low to moderate salinity (< 600 mM NaCl) and above pH 6. DIBMA-lipid nanoparticles could be formed above a pH of 5 and were stable in up to 4 M NaCl. Similarly, PMA-lipid nanoparticles were stable in all NaCl concentrations tested (up to 4 M) and a broad pH range (3-10). However, for both DIBMA and PMA nanoparticles there is a severe penalty observed for bilayer solubilisation in non-optimal conditions or when using a charged membrane. Additionally, lipid fluidity of the DMPC-polymer nanoparticles was analysed through cw-EPR, showing no cooperative gel-fluid transition as would be expected for native-like lipid membranes.

Quantification of nitrogen cycle functional genes from viable archaea and bacteria in paddy soil.

AIMS: One of the main challenges of culture-independent soil microbiology is distinguishing the microbial community's viable fraction from dead matter. Propidium monoazide (PMA) binds the DNA of dead cells, preventing its amplification. This dye could represent a robust means to overcome the drawbacks of other selective methods, such as ribonucleic acid-based analyses. METHODS AND RESULTS: We quantified functional genes from viable archaea and bacteria in soil by combining the use of PMA and quantitative polymerase chain reaction. Four N-cycle-related functional genes (bacterial and archaeal ammonia monooxygenase, nitrate reductase, and nitrite reductase) were successfully quantified from the living fraction of bacteria and archaea of a paddy soil. The protocol was also tested with pure bacterial cultures and soils with different physical and chemical properties. CONCLUSIONS: The experiment results revealed a contrasting impact of mineral and organic fertilizers on the abundance of microbial genes related to the N-cycle in paddy soil.

Quantification and molecular characterization of intact rotavirus species A (RVA) in municipal solid waste leachate.

AIMS: Leachate comprises a solid waste decomposition product found fresh in collection trucks or as an effluent in landfills. This study aimed to assess the occurrence, concentrations, and genetic diversity of intact rotavirus species A (RVA) in solid waste leachate. METHODS AND RESULTS: Leachate samples were concentrated by ultracentrifugation, treated with propidium monoazide (PMA), and exposed to LED photolysis. Treated and untread samples were extracted using the QIAamp Fast DNA Stool mini kit, and nucleic acids were screened for RVA employing a Taqman® Real-time PCR. The PMA RT-qPCR method detected RVA in eight out of nine truck samples and in 15.40% (2/13) of the landfill leachate samples. The RVA concentrations in the PMA-treated samples ranged from 4.57 × 103 to 2.15 × 107 genomic copies (GC) 100 mL-1 in truck leachate and from 7.83 × 103 to 1.42 × 104 GC 100 mL-1 in landfill samples. Six truck leachate samples were characterized as RVA VP6 genogroup I2 by partial nucleotide sequencing. CONCLUSIONS: The high intact RVA detection rates and concentrations in truck leachate samples indicate potential infectivity and comprise a warning for solid waste collectors concerning hand-to-mouth contact and the splash route.

Quickly assessing disinfection effectiveness to control the spread of African swine fever virus.

Infectious African swine fever virus (ASFV) can cause the spread and morbidity of African swine fever, while the inactivated virus cannot. When they are not distinguished separately, the detection results will lack authenticity and cause unnecessary panic and detection cost. The detection technology based on cell culture is complex, high-cost, and time-consuming in practice, which is not conducive to the rapid detection of infectious ASFV. In this study, a propidium monoazide (PMA) qPCR detection method for rapid diagnosis of infectious ASFV was constructed. Parameters of PMA concentration, light intensity, and lighting time were under strict safety verification and comparative analysis for optimization. The results determined that the optimal condition for PMA to pretreat ASFV was the final concentration of PMA 100 µM. The light intensity was 40 W, the light duration was 20 min, the target fragment size of the optimal primer probe was 484 bp, and its detection sensitivity for infectious ASFV was 101.28 HAD50/mL. In addition, the method was innovatively applied to the rapid evaluation of disinfection effect. When ASFV concentration was less than 102.28 HAD50/mL, the method could still be effective for the evaluation of thermal inactivation effect, and the evaluation ability of chlorine-containing disinfectants was better, and the applicable concentration could reach 105.28 HAD50/mL. It is worth mentioning that this method can not only reflect whether the virus is inactivated, but also indirectly reflect the degree of damage to viral nucleic acid caused by disinfectants. In conclusion, the PMA-qPCR constructed in this study can be applied to laboratory diagnosis, disinfection effect evaluation, drug development, and other aspects of infectious ASFV and can provide new technical support for effective prevention and control of ASF. KEY POINTS: • A rapid detection method for infectious ASFV was developed • Provide a new scheme for rapid evaluation of disinfection effect of chlorine-containing disinfectants • PMA-qPCR can simultaneously show the survival status of the virus and the damage of nucleic acid.

Hydrogen sulfide improves salt tolerance through persulfidation of PMA1 in Arabidopsis.

KEY MESSAGE: A new interaction was found between PMA1 and GRF4. H2S promotes the interaction through persulfidated Cys446 of PMA1. H2S activates PMA1 to maintain K+/Na+ homeostasis through persulfidation under salt stress. Plasma membrane H+-ATPase (PMA) is a transmembrane transporter responsible for pumping protons, and its contribution to salt resistance is indispensable in plants. Hydrogen sulfide (H2S), a small signaling gas molecule, plays the important roles in facilitating adaptation of plants to salt stress. However, how H2S regulates PMA activity remains largely unclear. Here, we show a possible original mechanism for H2S to regulate PMA activity. PMA1, a predominant member in the PMA family of Arabidopsis, has a non-conservative persulfidated cysteine (Cys) residue (Cys446), which is exposed on the surface of PMA1 and located in cation transporter/ATPase domain. A new interaction of PMA1 and GENERAL REGULATORY FACTOR 4 (GRF4, belongs to the 14-3-3 protein family) was found by chemical crosslinking coupled with mass spectrometry (CXMS) in vivo. H2S-mediated persulfidation promoted the binding of PMA1 to GRF4. Further studies showed that H2S enhanced instantaneous H+ efflux and maintained K+/Na+ homeostasis under salt stress. In light of these findings, we suggest that H2S promotes the binding of PMA1 to GRF4 through persulfidation, and then activating PMA, thus improving the salt tolerance of Arabidopsis.

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.

Protective role of Acinetobacter and Bacillus for Escherichia coli O157:H7 in biofilms against sodium hypochlorite and extracellular matrix-degrading enzymes.

Foodborne pathogenic bacteria in multi-species biofilms in food manufacturing facilities have been suspected to be the cause of cross-contamination leading to foodborne illness. We studied if cafeteria kitchen-associated bacterial isolates can have any protective effect on E. coli O157:H7 in biofilm against extracellular polymeric substances (EPS)-degrading enzymes and sodium hypochlorite. We investigated multi-species biofilm-forming ability and the efficacy of EPS-degrading enzymes using crystal violet assay. The susceptibility of E. coli O157:H7 to sodium hypochlorite (NaClO) was evaluated using propidium monoazide combined with quantitative PCR (PMA-qPCR). Then, a combined treatment with enzymes followed by NaClO was also tested. Most cafeteria kitchen isolates of Acinetobacter and Bacillus were able to form biofilms. Several of them showed a protective effect on E. coli O157:H7 against NaClO after forming multi-species biofilms, particularly in Acinetobacter. This protective effect on E. coli O157:H7 was also noticed after the enzyme or the combined treatment with NaClO. Our results give us an insight into the protective role of food-associated environmental bacteria for E. coli O157:H7 in biofilms against common sanitizers and warrant further study to develop effective control methods. Our study also highlights the importance of preventing contamination or biofilm formation by environmental microorganisms, eventually reducing foodborne illness.

Regulation of proteolysis in bovine cumulus cells with possible inclusion of proton pump activators.

The aim of this study was to reveal the effects of V-ATPase proton pump activation on lysosomal acidity and protein degradation in cultured cumulus cells. Cumulus cells from bovine ovaries were cultured in the presence of 10 and 50 µM doses of V-ATPase proton pump activators PIP2, PMA and DOG for 12 and 24 h. At the end of the culture period, the level of protein degradation was evaluated through DQ-Red-BSA analysis and the lysosomes were detected through a fluorescent probe. In addition, total and phosphorylated MAPK1/3 and AKT protein levels of cumulus cells were determined through Western blotting. PIP2 and PMA were shown to increase protein degradation and lysosomal acidity in cultured bovine cumulus cells, whereas DOG did not have any significant effects on these cells. Total and phosphorylated MAPK and AKT protein levels were higher in PIP2 and PMA groups compared with the control and DOG. It was concluded that particular proton pump activators can enhance protein degradation and lysosomal acidification in cultured bovine cumulus cells without having detrimental effects on cell signalling members required for cell viability and proper functioning. Due to the cellular interactions, increasing the lysosomal activity in cumulus cells in the culture environment could also affect the removal of protein aggregates in the oocytes. This strategy could be effective for improving in vitro maturation of the oocytes by providing proteostasis.

Proton export upregulates aerobic glycolysis.

INTRODUCTION: Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the "Warburg Effect." It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. RESULTS: To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. CONCLUSIONS: Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.