Microplate Lethality Assay to Determine the Efficacy of Commercial Sanitizers for Inactivation of Listeria Monocytogenes, Escherichia coli O157:H7 and Salmonella in Biofilms

The prevalence of biofilms in food industries has caused serious problems. Different micro-organisms have been found to cross contaminate product itself, equipment and processes in food industries. Though various physical and biological methods have been applied to eliminate biofilms in food industries, chemical methods are still the most common and cost effective ways of biofilm prevention.The objective of this study was to determine efficacy of commercial sanitizers for inactivation of L. monocytogenes, E. coli O157:H7 and Salmonella in biofilms. L. monocytogenes, E. coli O157:H7 and Salmonella spp. were grown in black 96-well microplates and incubated with a fluorescent substrate (5,6-CFDA) to assess the degree of adherence or determine relative fluorescence unit (RFU) values with the help of fluorescent plate reader. Secondly, 7-day old biofilms of the adherent strains were grown in 96-well clear microplates and incubated for an hour at 37º C with different concentrations of enzymes. The recovered cells were then enumerated by plating on TSA plates to evaluate detaching ability of enzymes. Lastly, the 7-day old biofilms were treated with commercial sanitizers at various concentrations for different time periods in 96-well microplates. The reduction in number of cells was quantified by enzymatic detachment and plate counts and qualitatively assessed via scanning electron microscopy (SEM). Repeated Measures (RM) One-Way ANOVA was carried out to see significant differences (p7 log CFU/ml of Salmonella Montevideo FSIS051 in just 2.5 minutes of treatment.Thus, the application of new sanitizers (based on combination of several components) followed by enzymatic treatment may be the best option to kill and remove dead biofilms in food processing facilities.Animal Scienc

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Molecular Mechanism of Bruton’s Tyrosine Kinase Activation by the HIV-1 Nef Virulence Factor

Antiretroviral therapy has significantly boosted the effort in the fight against HIV. However, a cure has still not been found. The shortcomings associated with antiretroviral therapy underscore the need for alternative approaches to eliminate the latent viral reservoirs. HIV-1 Nef represents an attractive drug target in this regard because of its role in immune escape of HIV- infected cells. I explored the mechanism of interaction between HIV-1 Nef and Bruton’s tyrosine kinase (BTK), a member of the TEC tyrosine kinase family previously shown to enhance the HIV-1 life cycle in cells of myeloid lineage. I discovered that HIV-1 Nef activates BTK by a unique mechanism dependent upon SH3-SH2-mediated dimerization. In a solution-based assay, complex formation with HIV-1 Nef increased the stability of the BTK SH3-SH2 dimer. A cell-based bimolecular fluorescence complementation assay also showed increased recruitment of the BTK dimer to the cell membrane in presence of Nef. Alanine substitution of Pro327 in BTK SH2 domain CD-loop reduced the stability of the BTK dimer in solution, and also reduced BTK homodimerization in cells. Introduction of the P327A mutation completely uncoupled BTK from Nef-mediated activation in a kinetic kinase assay, and also prevented BTK dimerization and activation in response to Nef in cells. The effect of the P327A mutation is consistent with molecular modeling studies which identified a novel interface between the SH2 CD loop and the SH3 domain, where Pro327 is predicted to stabilize a specific CD loop conformation for SH3 engagement. An analogous interaction was demonstrated previously by NMR for the related kinase ITK, which also contributes to the HIV life cycle in CD4 T cells. Remarkably, this mechanism of BTK activation through stabilization of SH3•SH2 interaction is distinct from the SH3 domain displacement mechanism previously described for Nef- mediated activation of SRC-family kinases, indicating that this viral protein has evolved two separate mechanisms for host cell tyrosine kinase activation. Molecular details of the Nef-driven BTK activation process may guide future drug targeting of this unique interaction. The requirement of HIV-1 Nef homodimerization in this model suggests that HIV-1 Nef dimerization can be targeted for potential therapeutic intervention

Efficacy of Commercial Sanitizers Used in Food Processing Facilities for Inactivation of Listeria monocytogenes, E. Coli O157:H7, and Salmonella Biofilms

Bacteria entrapped in biofilms are a source of recurring problems in food processing environments. We recently developed a robust, 7-day biofilm microplate protocol for creating biofilms with strongly adherent strains of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella serovars that could be used to examine the effectiveness of various commercial sanitizers. Listeria monocytogenes 99-38, E.coli O157:H7 F4546, and Salmonella Montevideo FSIS 051 were determined from prior studies to be good biofilm formers and could be recovered and enumerated from biofilms following treatment with trypsin. Extended biofilms were generated by cycles of growth and washing daily, for 7 days, to remove planktonic cells. We examined five different sanitizers (three used at two different concentrations) for efficacy against the three pathogenic biofilms. Quaternary ammonium chloride (QAC) and chlorine-based sanitizers were the least effective, showing partial inhibition of the various biofilms within 2 h (1–2 log reduction). The best performing sanitizer across all three pathogens was a combination of modified QAC, hydrogen peroxide, and diacetin which resulted in ~6–7 log reduction, reaching levels below our limit of detection (LOD) within 1–2.5 min. All treatments were performed in triplicate replication and analyzed by one way repeated measures analysis of variance (RM-ANOVA) to determine significant differences (p < 0.05) in the response to sanitizer treatment over time. Analysis of 7-day biofilms by scanning electron microscopy (SEM) suggests the involvement of extracellular polysaccharides with Salmonella and E. coli, which may make their biofilms more impervious to sanitizers than L. monocytogenes

"Strategiczna autonomia" Indii i wzmacnianie jej więzi z USA

Immediately after independence, India joined the Non-Aligned Movement (NAM), which allowed it to avoid having to take sides with either of the then-existing two political-military blocs led by the US and the USSR. India under Jawaharlal Nehru, guided in foreign policy by principles derived from the traditions of Indian civilization, was not even able to anticipate and prevent China's aggression in 1962. Nehru's idealistic approach adopted by the Congress Party failed to ensure the permanence of power, and in 2014 the conservative-nationalist Indian People's Party (Bharatiya Janata Party) won the election, taking India's sovereignty and national interest as its supreme political imperative. The purpose of this article is to explain how the BJP government is striking a balance between India's traditional NAM-based strategic culture and growing defense ties with the US in the Indo-Pacific region. Therefore, it was necessary to present the evolution of Indian politics from radical non-alignment, grounded in the traditional value of non-violence (ahinsa), to so-called strategic autonomy.Wkrótce po uzyskaniu niepodległości India przystała do Ruchu Państw Niezaangażowanych, który pozwalał na uniknięcie konieczności opowiadania się po stronie żadnego z istniejących wówczas dwóch bloków polityczno-militarnych, którym przewodziły USA i ZSRR. India pod rządami Jawaharlala Nehru, kierując się w polityce zagranicznej zasadami wynikającymi z tradycji indyjskiej cywilizacji, nie były nawet zdolne przewidzieć i zapobiec agresji Chin w 1962 roku. Idealistyczne podejście Nehru przyjęte przez Partię Kongresową nie zapewniło trwałości władzy i w 2014 roku wybory wygrała konserwatywno-nacjonalistyczna Indyjska Partia Ludowa (Bharatiya Janata Party), dla której suwerenność i interes państwowy Indii stanowią naczelne imperatywy polityczne. Celem niniejszego artykułu jest wyjaśnienie, w jaki sposób rząd BJP osiąga równowagę między tradycyjną kulturą strategiczną Indii opartą na niezaangażowaniu, a rosnącymi więzami obronnymi z USA w regionie Indo-Pacyfiku. Dlatego niezbędne było przedstawienie ewolucji indyjskiej polityki od radykalnego niezaangażowania, mającego swoje oparcie w tradycyjnej wartości jaką stanowi powstrzymywanie się od przemocy (ahinsa) do tzw. strategicznej autonomii

Optimization of a Microplate Assay for Generating Listeria Monocytogenes, E. Coli O157:H7, and Salmonella Biofilms and Enzymatic Recovery for Enumeration

Biofilms enable the persistence of pathogens in food processing environments. Sanitizing agents are needed that are effective against pathogens entrapped in biofilms that are more difficult to inactivate than planktonic cells that are displaced and found on equipment surfaces. We examined conditions to develop, analyze, and enumerate the enhanced biofilms of three different foodborne pathogens assisted by fluorescence adherence assay and enzymatic detachment. We compared three different isomeric forms of fluorescent substrates that are readily taken up by bacterial cells based on carboxy-fluorescein diacetate (5-CFDA, 5,6-CFDA, 5,6-CFDA, SE). Biofilm-forming strains of Escherichia coli O157:H7 F4546 and Salmonella Montevideo FSIS 051 were identified using a microplate fluorescence assay defined previously for L. monocytogenes. Adherence levels were determined by differences in relative fluorescence units (RFU) as well as recovered bacterial cells. Multiple hydrolytic enzymes were examined for each representative pathogen for the most suitable enzyme for detachment and enumeration to confirm adherence data obtained by fluorescence assay. Cultures were grown overnight in microplates, incubated, washed and replenished with fresh sterile growth medium; this cycle was repeated for seven consecutive days to enrich for robust biofilms. Treatments were performed in triplicate and compared by one-way analysis of variance (ANOVA) to determine significant differences (p < 0.05)

Assessment of Climate Change Impacts on the Water, Food, and Energy Sectors in Sittaung River Basin, Myanmar

The Sittaung river basin (SRB) remains one of the least studied basins of Myanmar in terms of the assessment of the impact of climate change. As several reservoirs already exist in the basin, much research is needed to understand how projected climate change impacts rainfall, temperature, flows, domestic and agricultural demands, and hydropower generation. Given the limitation in observed data on the ground, a combination of satellite-derived meteorological data and digital elevation data is used to generate inputs to a Water Evaluation and Planning (WEAP) model. Five CMIP5 GCMs are used in the WEAP to assess the impact of climate change on the water, food, and energy production of the SRB for the baseline (BL: 1985–2014), near future (NF: 2021–2050), and far future (FF: 2051–2080) periods. The results indicate that the average temperature and rainfall are likely to increase in the future for the SRB. December and January are expected to be drier and warmer, whereas rainy months are expected to be wetter and warmer in the future. The BL flows (1091 m3/s) are expected to increase by 7–10% during NF and by 16–19% during FF at the basin outlet. Meanwhile, the unmet domestic demand during BL (1.3 MCM) is expected to decrease further by approximately 50% in the future. However, the unmet agricultural demand (667 MCM) for food production is estimated to increase from the BL by 11–15% during NF and by 14–19% during FF. Similarly, the total energy generation of nine hydropower projects (4.12 million MWh) is expected to increase by 9–11% during NF and by 16–17% during FF. Thus, the riverine flows are expected to increase in the future, thus positively impacting the domestic and hydropower sectors, whereas the unmet demands in the agricultural sector likely remain unsatisfied. These results will help the water, agriculture, and energy sectors to develop strategies to maximize benefits and cope with the impacts of climate change in the near and long-term future

Characterization of the arginine kinase isoforms in Caenorhabditis elegans

Phosphagen kinases (PKs) are well-studied enzymes involved in energy homeostasis in a wide range of animal, protozoan, and even some bacterial species. Recent genome efforts have allowed comparative work on the PKs to extend beyond the biochemistry of individual proteins to the comparative cellular physiology and examining of the role of all PK family members in an organism. The sequencing of the Caenorhabditis elegans genome and availability of sophisticated genetic tools within that system affords the opportunity to conduct a detailed physiological analysis of the PKs from a well known invertebrate for comparison with the extensive work conducted on vertebrate systems. As a first step in this effort we have carried out a detailed molecular genetic and biochemical characterization of the PKs in C. elegans. Our results reveal that C. elegans has five PK genes encoding arginine kinases that range in catalytic efficiency (kcat/KMArg) from (3.1 ± 0.6) × 104 to (9 ± 4) × 105 M− 1 s− 1. This range is generally within the range seen for arginine kinases from a variety of species. Our molecular genetic and phylogenetic analysis reveals that the gene family has undergone extensive intron loss and gain within the suborder Rhabditina. In addition, within C. elegans we find evidence of gene duplication and loss. The analysis described here for the C. elegans AKs represents one of the most complete biochemical and molecular genetic analysis of a PK family within a genetically tractable invertebrate system and opens up the possibility of conducting detailed physiological comparisons with vertebrate systems using the sophisticated tools available with this model invertebrate system