Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

Unstable Expression and Thermal Instability of a Species-Specific Cell Surface Epitope Associated with a 66-Kilodalton Antigen Recognized by Monoclonal Antibody EM-7G1 within Serotypes of Listeria monocytogenes Grown in Nonselective and Selective Broths

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective Listeria enrichment broth (LEB). When cells were grown in Listeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used Listeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other Listeria spp. (Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100°C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.     

Rhamnolipid biosurfactants: evolutionary implications,applications and future prospects from untapped marine resource

Rhamnolipid-biosurfactants are known to be produced by the genus Pseudomonas, however recent literature reported that rhamnolipids (RLs) are distributed among diverse microbial genera. To integrate the evolutionary implications of rhamnosyl transferase among various groups of microorganisms, a comprehensive comparative motif analysis was performed amongst bacterial producers. Findings on new RL-producing microorganism is helpful from a biotechnological perspective and to replace infective P. aeruginosa strains which ultimately ensure industrially safe production of RLs. Halotolerant biosurfactants are required for efficient bioremediation of marine oil spills. An insight on the exploitation of marine microbes as the potential source of RL biosurfactants is highlighted in the present review. An economic production process, solid-state fermentation using agro-industrial and industrial waste would increase the scope of biosurfactants commercialization. Potential and prospective applications of RL-biosurfactants including hydrocarbon bioremediation, heavy metal removal, antibiofilm activity/biofilm disruption and greener synthesis of nanoparticles are highlighted in this review.     

Physiology of Ex Planta Nitrogenase Activity in Rhizobium japonicum

Thirty-nine wild-type strains of Rhizobium japonicum have been studied for their ability to synthesize nitrogenase ex planta in defined liquid media under microaerobic conditions. Twenty-one produced more than trace amounts of acetylene reduction activity, but only a few of these yielded high activity. The oxygen response curves were similar for most of the nitrogenase-positive strains. The strains derepressible for activity had several phenotypic characteristics different from non-derepressible strains. These included slower growth and lower oxygen consumption under microaerobic conditions and lower extracellular polysaccharide production. Extracellular polysaccharide production during growth on gluconate in every nitrogenase-positive strain assayed was lower under both aerobic and microaerobic conditions than the non-derepressible strains. These phenotypic characteristics may be representative of a genotype of a subspecies of R. japonicum. These studies were done in part to enlarge the base number of strains available for studies on the physiology, biochemistry, and genetics of nitrogen fixation.     

Senescence marker protein 30 (SMP30) expression in eukaryotic cells: existence of multiple species and membrane localization

Senescence marker protein (SMP30), also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca(2+) homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA). Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ～28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of β and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.     

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: The case of chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD’s pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin—chelerythrine—acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.     

A model of stress and strain in the interosseous ligament of the forearm based on fiber network theory

Fiber network theory was developed to describe cloth, a thin material with strength in the fiber directions. The interosseous ligament (IOL) of the forearm is a broad, thin ligament with highly aligned fibers. The objectives of this study were to develop a model of the stress and strain distributions in the IOL, based on fiber network theory, to compare the strains from the model with the experimentally measured strains, and to evaluate the force distribution across the ligament fibers from the model. The geometries of the radius, ulna, and IOL were reconstructed from CT scans. Position and orientation of IOL insertion sites and force in the IOL were measured during a forearm compression experiment in pronation, neutral rotation, and supination. An optical image-based technique was used to directly measure strain in two regions of the IOL in neutral rotation. For the network model, the IOL was represented as a parametric ruled three-dimensional surface, with rulings along local fiber directions. Fiber strains were calculated from the deformation field, and fiber stresses were calculated from the strains using average IOL tensile properties from a previous study. The in situ strain in the IOL was assumed uniform and was calculated so that the net force predicted by the network model in neutral rotation matched the experimental result. The net force in the IOL was comparable to experimental results in supination and pronation. The model predicted higher stress and strain in fibers near the elbow in neutral rotation, and higher stresses in fibers near the wrist in supination. Strains in neutral forearm rotation followed the same trends as those measured experimentally. In this study, a model of stress and strain in the IOL utilizing fiber network theory was successfully implemented. The model illustrates variations in the stress and strain distribution in the IOL. This model can be used to show surgeons how different fibers are taut in different forearm rotation positions-this information is important for understanding the biomechanical role of the IOL and for planning an IOL reconstruction.