Some entomopathogenic fungi such as Metarhizium and Beauveria not only have the ability to infect and kill insects but also the capability to associate with plant roots. The potential benefits from ...this plant-fungus association include nutrient acquisition, plant growth promotion and protection against stressors. The objective of this study was to evaluate the delivery of entomopathogenic fungi in seed coating to determine whether the fungal presence affected maize performance. Additionally, fungal biocontrol ability was assessed in terms of plant resistance to the larvae of Costelytra giveni (Coleoptera: Scarabaeidae) and to the fungus Fusarium graminearum (Nectriaceae). Maize seeds were coated with conidia from Metarhizium spp. or Beauveria bassiana and plant performance was evaluated as seed germination and plant dry weight. Larval mortality and the presence of Fusarium root rot symptoms were also determined. The entomopathogenic fungal persistence on the maize rhizosphere was demonstrated with a M. anisopliae isolate expressing the green fluorescent protein. Presence of both challengers was detrimental to maize performance with 33% reduction in root dry weight in control plants while no variation was observed when the entomopathogenic fungi were used to coat seeds. Some seed coatings resulted in up to 67% mycosis of C. giveni larvae and a reduction in Fusarium rot root symptoms between 24 - 44%. This study showed that seed coating with conidia of Metarhizium or Beauveria can be used as a delivery system for pests and plant pathogen control, while at the same time hyphae formed a close association with plant roots after conidial germination.
Mesenchymal stromal cells (MSCs) may be of benefit in ARDS due to immunomodulatory and reparative properties. This trial investigates a novel CD362 enriched umbilical cord derived MSC product ...(REALIST ORBCEL-C), produced to Good Manufacturing Practice standards, in patients with moderate to severe ARDS due to COVID-19 and ARDS due to other causes.
Phase 1 is a multicentre open-label dose-escalation pilot trial. Patients will receive a single infusion of REALIST ORBCEL-C (100 × 10
cells, 200 × 10
cells or 400 × 10
cells) in a 3 + 3 design. Phase 2 is a multicentre randomised, triple blind, allocation concealed placebo-controlled trial. Two cohorts of patients, with ARDS due to COVID-19 or ARDS due to other causes, will be recruited and randomised 1:1 to receive either a single infusion of REALIST ORBCEL-C (400 × 10
cells or maximal tolerated dose in phase 1) or placebo. Planned recruitment to each cohort is 60 patients. The primary safety outcome is the incidence of serious adverse events. The primary efficacy outcome is oxygenation index at day 7. The trial will be reported according to the Consolidated Standards for Reporting Trials (CONSORT 2010) statement.
The development and manufacture of an advanced therapy medicinal product to Good Manufacturing Practice standards within NHS infrastructure are discussed, including challenges encountered during the early stages of trial set up. The rationale to include a separate cohort of patients with ARDS due to COVID-19 in phase 2 of the trial is outlined.
ClinicalTrials.gov NCT03042143. Registered on 3 February 2017. EudraCT Number 2017-000584-33.
An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F
420
. Studies have shown that actinobacterial F
420
H
2
-dependent reductases promiscuously hydrogenate diverse ...organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium
Mycobacterium smegmatis
. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F
420
H
2
. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F
420
H
-
to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F
420
H
-
) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F
420
H
2
-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.
The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, ...catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its kcat/Km value compared with that of the wild-type enzyme. The reduction in Km observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.
F
420
is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This ...deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F
420
(two glutamates) from a methanogen isolate and long-chain F
420
(five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F
420
purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F
420
-dependent oxidoreductases. However, long-chain F
420
bound to these enzymes with a six- to ten-fold higher affinity than short-chain F
420
. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F
420
increasing the substrate affinity (lower
K
m
) but reducing the turnover rate (lower
k
cat
) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F
420
makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F
420
to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F
420
will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F
420
-dependent oxidoreductases.
Hematological deficiencies increase with aging, including anemias, reduced responses to hematopoietic stress and myelodysplasias. This investigation tested the hypothesis that increased bone marrow ...(BM) fat content in humans with age was associated with decreased numbers of side population (SP) hematopoietic stem cells, and this decrease correlated with changes in cytokine levels. BM was obtained from the femoral head and trochanteric region of the femur removed at surgery for total hip replacement (N = 100 subjects). In addition, BM from cadavers (N = 36), with no evidence of hip disease, was evaluated for fat content. Whole trabecular marrow samples were ground in a sterile mortar and pestle, and cellularity and lipid content determined. Marrow cells were stained with Hoechst dye and SP profiles were acquired. Plasma levels of insulin‐like growth factor (IGF)‐1, stromal‐derived factor (SDF)‐1 and interleukin (IL)‐6 were measured using ELISA. Fat content in the BM of human subjects and cadavers increased with age. The numbers of SP stem cells in BM as well as plasma IGF‐1 and SDF‐1 levels decreased in correlation with increased BM fat. IL‐6 had no relationship to changes in marrow fat. These data suggest that increased BM fat may be associated with a decreased number of SP stem cells and IGF‐1 and SDF‐1 levels with aging. These data further raise a more general question as to the role of adipose cells in the regulation of tissue stem cells.
The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of ...this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.
A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F
This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including ...after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F
enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium
Mutant strains unable to synthesize or reduce F
were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F
mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F
H
-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover,
strains unable to make F
H
lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F
loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F
enhances antimicrobial resistance in mycobacteria and suggest that one function of the F
H
-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.
This study reveals that a unique microbial cofactor, F
, is critical for antimicrobial resistance in the environmental actinobacterium
We show that a superfamily of redox enzymes, the F
H
-dependent reductases, can reduce diverse antimicrobials
and
strains unable to make or reduce F
become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F
to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F
H
-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.
A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420 This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including ...after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium Mycobacterium smegmatis Mutant strains unable to synthesize or reduce F420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420 mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F420H2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCE This study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacterium Mycobacterium smegmatis We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobials in vitro and in vivoM. smegmatis strains unable to make or reduce F420 become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420 to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.
This study tests the hypothesis that hypocontractile, borderzone myocardium adjacent to an expanding infarct becomes progressively larger and more hypocontractile as remodeling continues.
Early ...infarct expansion following anteroapical myocardial infarction (MI) is associated with progressive ventricular dilation and heart failure. The contribution of perfused, hypocontractile, borderzone myocardium to this process is unknown.
Using a sheep model of anteroapical infarction, sonomicrometry array localization and serial microsphere injections were used to track changes in regional myocardial contractility, geometry, and perfusion. Eight sheep were studied before and after infarction and two, five, and eight weeks later. Thirty intertransducer chord lengths were analyzed to measure regional contractility and serial changes in regional geometry at end systole.
Beginning as a narrow band of fully perfused hypocontractile myocardium adjacent to the infarction, borderzone myocardium extends to involve additional contiguous myocardium that progressively loses contractile function as the heart remodels. Three distinct myocardial zones develop as a result of transmural MI: infarct, borderzone (perfused but hypocontractile), and remote (perfused and normally functioning).
This study demonstrates that hypocontractile, fully perfused borderzone myocardium extends to involve contiguous normal myocardium during postinfarction remodeling. This borderzone myocardium is a unique type of perfused, hypocontractile myocardium, which is distinct from hibernating or stunned myocardium. Preventing extension of borderzone myocardium by medical or surgical means offers the prospect of preventing late-onset heart failure following transmural expanding MIs.
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