scholarly journals First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors inKlebsiella pneumoniae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Dhruvitkumar S. Sutaria ◽  
Bartolome Moya ◽  
Kari B. Green ◽  
Tae Hwan Kim ◽  
Xun Tao ◽  
...  
Keyword(s):  
Rational Design ◽  
Binding Affinities ◽  
Content Type ◽  
Design And Optimization ◽  
High Affinity ◽  
Occupancy Patterns ◽  
Low Concentrations ◽  
Binding Data ◽  
Protein Occupancy ◽  
First Time

ABSTRACTPenicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium againstKlebsiella pneumoniae, PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors inK. pneumoniae. PBP binding was determined using isolated membrane fractions fromK. pneumoniaestrains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC50]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC50, <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC50, <0.0075 mg/liter) and avibactam (IC50, 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC50, 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC50, 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC50, 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors inK. pneumoniaeenable, for the first time, the rational design and optimization of double β-lactam and β-lactam–β-lactamase inhibitor combinations.

scholarly journals Cryo-electron Microscopy Structure and Transport Mechanism of a Wall Teichoic Acid ABC Transporter

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Li Chen ◽  
Wen-Tao Hou ◽  
Tao Fan ◽  
Banghui Liu ◽  
Ting Pan ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Abc Transporters ◽  
Rational Design ◽  
Teichoic Acid ◽  
Inhibitory Mechanism ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Design And Optimization ◽  
Cryo Electron Microscopy

ABSTRACT The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a “crankshaft conrod” mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA. IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a “crankshaft conrod” mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.

Design and optimization of a slot antenna for microwave heating of agglomerates

2018 ◽  
Vol 37 (6) ◽  
pp. 1886-1892
Author(s):  
Takahiro Kinoshita ◽  
Atsushi Sugihashi
Keyword(s):  
Microwave Heating ◽  
Radiation Efficiency ◽  
Field Analysis ◽  
Slot Antenna ◽  
Productivity Improvement ◽  
Crushing Strength ◽  
Content Type ◽  
Design And Optimization ◽  
First Time ◽  
Antenna Position

Purpose The microwave heating technique can be used in the production of iron for productivity improvement when the consumption of low-quality iron ore increases. Iron-containing dust agglomerates are an important source of iron; however, the crushing strength of raw agglomerates is low, which decreases the yield owing to breaking. Therefore, they need to be dried using a “band dryer” to reduce the amount of excess water and improve their crushing strength. This paper aims to present the structure of an antenna that efficiently provides microwave energy to the agglomerate layer inside the band dryer. Design/methodology/approach Electromagnetic field analysis and experiments that simulate inside the band dryer were performed to evaluate the radiation efficiency of the slot antenna for microwave heating of the agglomerate layer in the presence of a metal conveyor. Findings The results show that slot height should be larger than the antenna position from the metal conveyor and smaller than the sum of the antenna position and quarter of the guide wavelength, to maximize the radiation efficiency of the antenna. By satisfying this condition, the radiation efficiency was higher than 75 per cent in the experiments. Originality/value This paper presents for the first time the optimum relation between slot height and antenna position to apply the microwave heating technique to iron-containing agglomerates in a band dryer.

scholarly journals Utility of Immunoblotting for Early Diagnosis of Toxoplasmosis Seroconversion in Pregnant Women

2011 ◽  
Vol 18 (11) ◽  
pp. 1908-1912 ◽  
Author(s):  
C. Jost ◽  
F. Touafek ◽  
A. Fekkar ◽  
R. Courtin ◽  
M. Ribeiro ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Transmission Rate ◽  
Immunoblot Analysis ◽  
Immunofluorescence Assay ◽  
Serum Samples ◽  
Content Type ◽  
Low Concentrations ◽  
Qualitative Test ◽  
First Time

ABSTRACTCongenital transmission ofToxoplasma gondiioccurs mainly when a mother acquires the infection for the first time during pregnancy. It was recently shown that although early treatment of the primary infection during pregnancy has little or no impact on the fetomaternal transmission rate, it does reduce the incidence of sequelae in infected infants. Seroconversion is defined by the appearance of IgG. Commercial reagents continue to vary considerably in detecting low concentrations of antibodies, as during early seroconversion. We compared two routinely used immunoassays (IA) (Platelia and Elecsys Toxo IgG) and an indirect immunofluorescence assay (IIF) with a qualitative test based on immunoblot analysis (Toxo II IgG) (IB) to assess their abilities to diagnose seroconversion at its earliest stages. This prospective study was carried out between January and November 2010. It included 39 pregnant women with monthly follow-up who seroconverted during pregnancy. On first sera that were IgM positive but IgG negative (or equivocal) as detected by IA, IB diagnosed seroconversion twice as often as IIF (26/39 [66.7%] versus 13/39 [33.3%];P< 0.001; χ2test). Serum samples were retaken 2 to 5 weeks later for the other 13 cases (IgG negative by IB on first serum). Seroconversion was demonstrated as follows: IB for 5 cases where IA remained negative or equivocal, IB and IIF for 5 cases where IA remained negative or equivocal, IA for 2 cases, and no method for 1 case (a third sample was necessary). In summary, IB permitted toxoplasmosis seroconversion diagnosis before other means in 92.3% of cases (36/39) and thus earlier therapeutic intervention.

scholarly journals First Penicillin-Binding Protein Occupancy Patterns for 15 β-Lactams and β-Lactamase Inhibitors in Mycobacterium abscessus

2020 ◽  
Vol 65 (1) ◽  
pp. e01956-20
Author(s):  
Alaa R. M. Sayed ◽  
Nirav R. Shah ◽  
Kari B. Basso ◽  
Manasi Kamat ◽  
Yuanyuan Jiao ◽  
...  
Keyword(s):  
Binding Protein ◽  
Principal Component ◽  
Mycobacterium Abscessus ◽  
Success Rates ◽  
Penicillin Binding Protein ◽  
Standard Regimen ◽  
Content Type ◽  
Occupancy Patterns ◽  
Low Concentrations ◽  
Effective Combination

ABSTRACTMycobacterium abscessus causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of M. abscessus infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With β-lactam antibiotics being safe, double β-lactam and β-lactam/β-lactamase inhibitor combinations are of interest for improving the treatment of M. abscessus infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different β-lactams in M. abscessus. We determined the preferred PBP targets of 13 β-lactams and 2 β-lactamase inhibitors in two M. abscessus strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the β-lactam concentrations that half-maximally inhibited Bocillin binding (50% inhibitory concentrations [IC50s]). Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016 to 0.5 mg/liter) (cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031 to 1 mg/liter) (ceftriaxone and cefotaxime) or intermediate (0.35 to 16 mg/liter) (ceftazidime and cefoxitin) concentrations. Sulbactam, aztreonam, carumonam, mecillinam, and avibactam (cluster 3) inactivated the same PBPs as cephalosporins but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2 to 16 mg/liter. Carbapenems, ceftriaxone, and cefotaxime were the most promising β-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in M. abscessus provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with β-lactams.

scholarly journals β-Lactam Antibiotics with a High Affinity for PBP2 Act Synergistically with the FtsZ-Targeting Agent TXA707 against Methicillin-Resistant Staphylococcus aureus

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Edgar Ferrer-González ◽  
Malvika Kaul ◽  
Ajit K. Parhi ◽  
Edmond J. LaVoie ◽  
Daniel S. Pilch
Keyword(s):  
Staphylococcus Aureus ◽  
Significant Risk ◽  
Standard Of Care ◽  
Multidrug Resistant ◽  
Binding Affinities ◽  
Current Standard ◽  
Methicillin Resistant ◽  
Content Type ◽  
High Affinity ◽  
Fluorescence And Electron Microscopy

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. We have developed a promising new FtsZ-targeting agent (TXA707) with potent activity against MRSA isolates resistant to current standard-of-care antibiotics. We present here results that demonstrate differing extents of synergy between TXA707 and a broad range of β-lactam antibiotics (including six cephalosporins, two penicillins, and two carbapenems) against MRSA. To explore whether there is a correlation between the extent of synergy and the preferential antibacterial target of each β-lactam, we determined the binding affinities of the β-lactam antibiotics for each of the four native penicillin-binding proteins (PBPs) of S. aureus using a fluorescence anisotropy competition assay. A comparison of the resulting PBP binding affinities with our corresponding synergy results reveals that β-lactams with a high affinity for PBP2 afford the greatest degree of synergy with TXA707 against MRSA. In addition, we present fluorescence and electron microscopy studies that suggest a potential mechanism underlying the synergy between TXA707 and the β-lactam antibiotics. In this connection, our microscopy results show a disruption of septum formation in TXA707-treated MRSA cells, with a concomitant mislocalization of the PBPs from midcell to nonproductive peripheral sites. Viewed as a whole, our results indicate that PBP2-targeting β-lactam antibiotics are optimal synergistic partners with FtsZ-targeting agents for use in combination therapy of MRSA infections.

The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

1978 ◽  
Vol 40 (02) ◽  
pp. 350-357
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Molecular Weight ◽  
Dissociation Constant ◽  
Calcium Ions ◽  
Activation Process ◽  
Factor X ◽  
The Real ◽  
High Affinity ◽  
Binding Data ◽  
Sequential Mechanism ◽  
Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

Formation and Trapping of the Thermodynamically Unfavoured Inverted-Hemicucurbit[6]uril

2019 ◽  
Author(s):  
Elena Prigorchenko ◽  
Sandra Kaabel ◽  
Triin Narva ◽  
Anastassia Baškir ◽  
Maria Fomitšenko ◽  
...  
Keyword(s):  
Complex Formation ◽  
Combinatorial Chemistry ◽  
Trifluoroacetic Acid ◽  
Chloride Anion ◽  
Binding Affinities ◽  
Dynamic Covalent Chemistry ◽  
Reaction Selectivity ◽  
Low Concentration ◽  
Dynamic Combinatorial Chemistry ◽  
First Time

Amplification of a thermodynamically unfavoured macrocyclic product through the directed shift of the equilibrium between dynamic covalent chemistry library members is difficult to achieve. We show for the first time that during condensation of formaldehyde and <i>cis</i>-<i>N,N'</i>-cyclohexa-1,2-diylurea formation of <i>inverted-cis</i>-cyclohexanohemicucurbit[6]uril (<i>i-cis</i>-cycHC[6]) can be induced at the expense of thermodynamically favoured <i>cis</i>-cyclohexanohemicucurbit[6]uril (<i>cis</i>-cycHC[6]). The formation of <i>i-cis-</i>cycHC[6] is enhanced in low concentration of the templating chloride anion and suppressed in excess of this template. We found that reaction selectivity is governed by the solution-based template-aided dynamic combinatorial chemistry and continuous removal of the formed cycHC[6] macrocycles from the equilibrating solution by precipitation. Notably, the <i>i-cis</i>-cycHC[6] was isolated with 33% yield. Different binding affinities of three diastereomeric <i>i-cis</i>-, <i>cis</i>-cycHC[6] and their chiral isomer (<i>R,R</i>)-cycHC[6] for trifluoroacetic acid demonstrate the influence of macrocycle geometry on complex formation.

Conference report: innovation, interaction … and a lot of data: first time reflections on Internet Librarian International, UK 2018

2019 ◽  
Vol 36 (2) ◽  
pp. 21-22
Author(s):  
Ray Harper
Keyword(s):  
Best Practice ◽  
New Technologies ◽  
Conference Report ◽  
Library Services ◽  
New Techniques ◽  
Content Type ◽  
Making A Difference ◽  
Analyse Data ◽  
Physical Spaces ◽  
First Time

Purpose The purpose of this paper is to summarise a number of presentations at Day 1 of the Internet Librarian International conference, London, UK (16 October 2018). This was the 20th conference in the series, and the three key themes included were the next-gen library and librarian; understanding users, usage and user experience; and inclusion and inspiration: libraries making a difference. Design/methodology/approach This paper reports from the viewpoint of a first-time attendee of the conference. This summarises the main issues raised by each presentation and draws out the key learning points for practical situations. Findings The conference covered a variety of practical ways in which libraries can use technology to support users and make decisions about services. These include developing interactive physical spaces which include augmented reality; introducing “chat-bots” to support users; using new techniques to analyse data; and piloting new ways to engage users (such as coding clubs). A key theme was how we use and harness data in a way that is ethical, effective and relevant to library services. Originality/value This conference focussed on practical examples of how library and information services across sectors and countries are innovating in a period of huge change. The conference gave delegates numerous useful ideas and examples of best practice and demonstrated the strength of the profession in adapting to new technologies and developments.

Interest rate option hedging portfolios without bank account

2019 ◽  
Vol 37 (1) ◽  
pp. 134-142
Author(s):  
Alberto Bueno-Guerrero
Keyword(s):  
Design Methodology ◽  
Contingent Claim ◽  
Necessary And Sufficient Condition ◽  
Barrier Option ◽  
Bank Account ◽  
Content Type ◽  
Option Hedging ◽  
Hedging Portfolio ◽  
Necessary And Sufficient ◽  
First Time

Purpose This paper aims to study the conditions for the hedging portfolio of any contingent claim on bonds to have no bank account part. Design/methodology/approach Hedging and Malliavin calculus techniques recently developed under a stochastic string framework are applied. Findings A necessary and sufficient condition for the hedging portfolio to have no bank account part is found. This condition is applied to a barrier option, and an example of a contingent claim whose hedging portfolio has a bank account part different from zero is provided. Originality/value To the best of the authors’ knowledge, this is the first time that this issue has been addressed in the literature.

A “Learning from Action” workshop in Japan

2019 ◽  
Vol 40 (2) ◽  
pp. 77-83
Author(s):  
Robert Douglas Hinshelwood ◽  
Luca Mingarelli ◽  
Simona Masnata
Keyword(s):  
Far East ◽  
Opportunity To Learn ◽  
Content Type ◽  
Work Activities ◽  
Defensive Role ◽  
Feedback Data ◽  
Experiential Workshop ◽  
First Time ◽  
Practical Implications

Purpose Many people in severe mentally disturbed states do not use language or other symbolic media well or coherently. Therefore, the non-verbal medium needs to be understood by workers with such people. The “Learning from Action” experiential workshop was developed in order to provide an opportunity to learn about hidden messages in the relationships and roles occurring in activities. In August 2017, a workshop was run for the first time in Japan. The purpose of this paper is to report the experience and dynamics observed by the three consultants, who are here the authors of this paper. Design/methodology/approach After the workshop all the staff and members, including interpreters, were invited to give feedback. Findings Analysis of the feedback data showed certain important dynamics, concerning especially dependence, cultural defences and the defensive role of activity in a multicultural context. Research limitations/implications This is an initial experience to be followed up by later feedback and further workshops. Practical implications Workers awareness of non-verbal communication within the roles of work activities is a training possibility. It faces various resistances including the mental health assumptions of meaninglessness of any communication outside the verbal. Originality/value This is a method of training not widely used even in European countries, and is the first in a country in the far east.

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