Characterization of mutants with single and combined Qi and Qo site mutations in Saccharomyces cerevisiae reveals interactions between the picolinamide fungicide CAS-649 and azoxystrobin.

Picolinamide and strobilurin fungicides bind to the Qi and Qo sites on cytochrome b, respectively, and target many of the same plant pathogens. Using Saccharomyces cerevisiae as a model system, we explore effects of amino acid changes at each site on sensitivity to a fungicide acting at the opposite site and examine the relationship between altered sensitivity and growth penalty. In addition, double mutants containing the G143A or F129L mutations responsible for strobilurin resistance in combination with Qi site mutations that confer resistance to picolinamides are characterized in terms of their sensitivity to QiI and QoI fungicides and growth rate. Mutants containing amino acid changes at the Qo site varied in their growth rate and sensitivity to the picolinamide CAS-649, and increased sensitivity was associated with a greater growth penalty. Conversely, changes at the Qi site affected sensitivity to azoxystrobin and also showed a correlation between increased sensitivity and reduced growth. There was no overall correlation between resistance to azoxystrobin and CAS-649 among mutants, however negative cross-resistance occurred in the case of mutations which conferred resistance to either compound and also carried a growth penalty. These results suggest the use of QoI fungicides to delay the emergence of pathogen resistance to QiIs, and vice versa. Double mutants containing G143A or F129L in combination with Qi site changes N31K, G37C/V or L198F that cause resistance to picolinamides generally exhibited lower resistance factors for both azoxystrobin and CAS-649 than corresponding resistant strains with a single mutation. Reduced growth was observed for all F129L-containing double mutants, whereas the growth rate of double mutants containing G143A was significantly reduced only by the Qi site mutations N31K and G37V that confer a larger growth penalty. Our results suggest that resistance to picolinamides in pathogens could emerge more readily in a strobilurin-sensitive genetic background than in a strobilurin-resistant one.

Interaction of picolinamide fungicide primary metabolites UK-2A and CAS-649 with the cytochrome bc1 complex Qi site: mutation effects and modelling in Saccharomyces cerevisiae.

BACKGROUND: Fenpicoxamid and florylpicoxamid are picolinamide fungicides targeting the Qi site of the cytochrome bc1 complex, via their primary metabolites UK-2A and CAS-649, respectively. We explore binding interactions and resistance mechanisms for picolinamides, antimycin A and ilicicolin H in yeast by testing effects of cytochrome b amino acid changes on fungicide sensitivity and interpreting results using molecular docking. RESULTS: Effects of amino acid changes on sensitivity to UK-2A and CAS-649 were similar, with highest resistance associated with exchanges involving G37 and substitutions N31K and L198F. These changes, as well as K228M, also affected antimycin A, while ilicicolin H was affected by changes at G37 and L198, as well as Q22E. N31 substitution patterns suggest that a lysine at position 31 introduces an electrostatic interaction with neighbouring D229, causing disruption of a key salt-bridge interaction with picolinamides. Changes involving G37 and L198 imply resistance primarily through steric interference. G37 changes also showed differences between CAS-649 and UK-2A or antimycin A with respect to branched versus unbranched amino acids. N31K and substitution of G37 by large amino acids reduced growth rate substantially while L198 substitutions showed little effect on growth. CONCLUSION: Binding of UK-2A and CAS-649 at the Qi site involves similar interactions such that general cross-resistance between fenpicoxamid and florylpicoxamid is anticipated in target pathogens. Some resistance mutations reduced growth rate and could carry a fitness penalty in pathogens. However, certain changes involving G37 and L198 carry little or no growth penalty and may pose the greatest risk for resistance development in the field. © 2022 Society of Chemical Industry.

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici.

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Qo site). We identified the G37V change within the cytochrome b Qi site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Qo site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.

Nutritional Assessment: A Primary Component of the Multidimensional Geriatric Assessment in the Intensive Care Unit.

The importance of evaluating and adjusting the nutritional state of critically ill patients has become a core principle of care. This article focuses on tools for the nutritional assessment of geriatric intensive care unit patients, including a review of imaging and other standardized techniques for evaluation of muscle mass, an indicator of malnutrition and sarcopenia. It concludes with a discussion of the interplay of malnutrition, reduced muscle mass/sarcopenia, and frailty. The goal of this multidimensional assessment is to identify those at risk and thereby initiate interventions to improve outcomes.

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK-2A.

BACKGROUND: Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK-2A. Its mode of action and target site interactions have been investigated. RESULTS: UK-2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK-2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK-2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK-2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross-resistance to strobilurin, azole or benzimidazole fungicides. CONCLUSION: Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK-2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transfusion-associated graft versus host disease in the immunocompetent patient: an ongoing problem.

Transfusion associated-graft versus host disease (TA-GVHD) is a rare complication of blood transfusion. It carries a very high mortality rate. Although the phenomenon has been well described in immunocompromised patients, this review focuses on the immunocompetent host. Cases of TA-GVHD continue to be reported following a variety of surgical procedures, especially cardiac procedures requiring cardiopulmonary bypass. Additional risk factors for TA-GVHD include blood component transfusion in populations with limited genetic diversity, the use of directed donations from family members, and the transfusion of fresh blood. As there is no effective treatment, the focus is on prevention.

Alcohol withdrawal syndrome in admitted trauma patients.

BACKGROUND: As alcohol use is highly prevalent in trauma patients, we hypothesized that a significant proportion of hospitalized trauma patients would demonstrate alcohol withdrawal (AW). METHODS: The trauma registries at a joint trauma center system from 1999 to 2008 were evaluated for patients aged at least 16 years. RESULTS: Of 19,369 trauma admissions, 159 patients had AW. Blood alcohol concentration (BAC) testing was performed in 31.5% of the patients. BAC was significantly higher in AW patients versus other traumas (205.7 ± 130.1 vs 102.9 ± 121.7 mg/dL). BAC was 0 in 14.4% of AW patients. As compared with other trauma patients, patients with AW had a significantly greater age (50.2 vs 42.1 years), hospital length of stay (10 vs 3 days), intensive care unit length of stay (2 vs 0 days), need for mechanical ventilation (34% vs 12.7%), and pneumonia (12% vs 2.3%). AW patients were less frequently discharged to home (59.8% vs 69.9%). Mortality was not different. CONCLUSIONS: AW was diagnosed in few patients. Of note, it occurred in patients with an initial BAC of 0. AW is associated with adverse outcomes.

Farm machinery injuries: the 15-year experience at an urban joint trauma center system in a rural state.

Farm machinery is a major source of injury. The objective of this study is to characterize the incidence, injury characteristics, and outcomes of patients admitted with farm machinery injuries (FMIs) to an urban joint trauma system in a rural state. A retrospective 15-year review of the trauma registries of the two trauma centers that function as a single state-designated Level I joint trauma center system was conducted. There were 65 admissions for FMIs at hospital A and 41 at hospital B; this represents under 0.4% of total trauma admissions. The patients ranged in age from 2 to 87 years. At hospital A, 89% of admitted patients sustained extremity injuries, 16% sustained torso trauma, 92% required surgical intervention, and the mortality rate was 0%. At hospital B, 60% of admitted patients sustained extremity injuries, 36.6% of patients sustained torso trauma, 63% required surgical intervention, and the mortality rate was 14.6%. Tractor-related injuries were responsible for 17% of admissions at hospital A and 69% at hospital B. Of the six fatalities, five were tractor related. The data demonstrate that FMIs affect people in nearly all decades of life. FMIs at the two hospitals had differing injury characteristics and outcomes, in large part secondary to the differing frequency of tractor-related injuries. FMIs frequently required surgical intervention.

A quick primer for setting up and maintaining surgical intensive care in an austere environment: practical tips from volunteers in a mass disaster.

The provision of critical care in any environment is resource intensive. However, the provision of critical care in an austere environment/mass disaster zone is particularly challenging. While providers are well trained for care in a modern intensive care unit, they may be under-prepared for resource-poor environments where there are limited or unfamiliar equipment and fewer support personnel. Based primarily on our experiences at a field hospital in Haiti, we created a short guide to critical care in a mass disaster in an austere environment. This guide will be useful to the team of physicians, nurses, respiratory care, logistics, and other support personnel who volunteer in future critical care relief efforts in limited resource settings.

Two hospitals with 1 trauma system: a joint approach to the care of the injured patient.

BACKGROUND: Trauma centers are closing at an alarming rate, but the need for trauma care persists. This article shows the sustainability and feasibility of a joint trauma system whereby 2 university-affiliated hospitals function as a single trauma center system in a moderate-sized city. METHODS: Since 1994, 3 days per week, trauma patients are transported by emergency medical services (EMS) to hospital A. The other 4 days they are transported to hospital B. Trauma registry data from 1994 to 2008 were analyzed. Cost data were also examined. RESULTS: The joint system admitted 28,338 trauma patients. On each center's nontrauma days, trauma team activation was required infrequently. The 2 centers share costs; they perform joint outreach, educational training, and quality control. The joint trauma system has been sustained since 1994. CONCLUSIONS: Two hospitals functioning as a single trauma center system is a viable model of care for injured patients in a moderate-sized city with mostly blunt trauma.

A radioligand binding assay for antitubulin activity in tumor cells.

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.

Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores.

Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC(50)s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC(50)s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.