scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

Fused filament printing of specialized biomedical devices: a state-of-the art review of technological feasibilities with PEEK

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Erfan Rezvani Ghomi ◽  
Saeideh Kholghi Eshkalak ◽  
Sunpreet Singh ◽  
Amutha Chinnappan ◽  
Seeram Ramakrishna ◽  
...  
Keyword(s):  
High Performance ◽  
State Of The Art ◽  
Three Dimensional ◽  
Patient Specific ◽  
Biomedical Devices ◽  
Fused Filament Fabrication ◽  
Content Type ◽  
Patient Specific Implants ◽  
Complex Design ◽  
Wide Range

Purpose The potential implications of the three-dimensional printing (3DP) technology are growing enormously in the various health-care sectors, including surgical planning, manufacturing of patient-specific implants and developing anatomical models. Although a wide range of thermoplastic polymers are available as 3DP feedstock, yet obtaining biocompatible and structurally integrated biomedical devices is still challenging owing to various technical issues. Design/methodology/approach Polyether ether ketone (PEEK) is an organic and biocompatible compound material that is recently being used to fabricate complex design geometries and patient-specific implants through 3DP. However, the thermal and rheological features of PEEK make it difficult to process through the 3DP technologies, for instance, fused filament fabrication. The present review paper presents a state-of-the-art literature review of the 3DP of PEEK for potential biomedical applications. In particular, a special emphasis has been given on the existing technical hurdles and possible technological and processing solutions for improving the printability of PEEK. Findings The reviewed literature highlighted that there exist numerous scientific and technical means which can be adopted for improving the quality features of the 3D-printed PEEK-based biomedical structures. The discussed technological innovations will help the 3DP system to enhance the layer adhesion strength, structural stability, as well as enable the printing of high-performance thermoplastics. Originality/value The content of the present manuscript will motivate young scholars and senior scientists to work in exploring high-performance thermoplastics for 3DP applications.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

Hepatocyte Aggregate Culture Technique for Bioreactors in Hybrid Liver Support Systems

1993 ◽  
Vol 16 (12) ◽  
pp. 843-846 ◽  
Author(s):  
J.C. Gerlach ◽  
K. Klöppel ◽  
C. MÜller ◽  
N. Schnoy ◽  
M.D. Smith ◽  
...  
Keyword(s):  
High Performance ◽  
Large Scale ◽  
Support Systems ◽  
Three Dimensional ◽  
Culture Technique ◽  
Dimensional Structure ◽  
Cell Aggregates ◽  
Liver Support ◽  
Aggregate Culture

Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.

scholarly journals In Vitro Activity of Imipenem-Relebactam and Ceftolozane-Tazobactam against Resistant Gram-Negative Bacilli

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Suzannah M. Schmidt-Malan ◽  
Avisya J. Mishra ◽  
Ammara Mushtaq ◽  
Cassandra L. Brinkman ◽  
Robin Patel
Keyword(s):  
Antimicrobial Agents ◽  
Empirical Therapy ◽  
Drug Resistant ◽  
Rapid Diagnostics ◽  
In Vitro Activity ◽  
Gram Negative ◽  
Content Type ◽  
Inhibitor Combination ◽  
Selection Of

ABSTRACT Understanding which antimicrobial agents are likely to be active against Gram-negative bacilli can guide selection of antimicrobials for empirical therapy as mechanistic rapid diagnostics are adopted. In this study, we determined the MICs of a novel β-lactam–β-lactamase inhibitor combination, imipenem-relebactam, along with ceftolozane-tazobactam, imipenem, ertapenem, meropenem, ceftriaxone, and cefepime, against 282 drug-resistant isolates of Gram-negative bacilli. For isolates harboring blaKPC (n = 110), the addition of relebactam to imipenem lowered the MIC50/MIC90 from 16/>128 μg/ml for imipenem alone to 0.25/1 μg/ml. For isolates harboring blaCTX-M (n = 48), the MIC50/MIC90 of ceftolozane-tazobactam were 0.5/16 μg/ml (83% susceptible). For isolates harboring blaCMY-2 (n = 17), the MIC50/MIC90 of ceftolozane-tazobactam were 4/8 μg/ml (47% susceptible). Imipenem-relebactam was active against most KPC-producing (but not NDM- or IMP-producing) Enterobacteriaceae and is an encouraging addition to the present antibiotic repertoire.

scholarly journals Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
J. Wendler ◽  
D. Ehmann ◽  
L. Courth ◽  
B. O. Schroeder ◽  
N. P. Malek ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Outer Membrane ◽  
Specific Activity ◽  
Redox System ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Redox Systems ◽  
Native Form ◽  
Gram Negative ◽  
Content Type

ABSTRACTThe antimicrobial peptide human β-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants ofEscherichia colilacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane ofE. coli. However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

RBF-based mesh morphing approach to perform icing simulations in the aviation sector

2019 ◽  
Vol 91 (4) ◽  
pp. 620-633 ◽  
Author(s):  
Corrado Groth ◽  
Emiliano Costa ◽  
Marco Evangelos Biancolini
Keyword(s):  
High Performance ◽  
Three Dimensional ◽  
Ansys Fluent ◽  
Content Type ◽  
Mesh Morphing ◽  
Computational Fluid Dynamics Cfd ◽  
Cfd Analyses ◽  
2D And 3D ◽  
First Time ◽  
Practical Implications

Purpose Numerical simulation of icing has become a standard. Once the iced shape is known, however, the analyst needs to update the computational fluid dynamics (CFD) grid. This paper aims to propose a method to update the numerical mesh with ice profiles. Design/methodology/approach The present paper concerns a novel and fast radial basis functions (RBF) mesh morphing technique to efficiently and accurately perform ice accretion simulations on industrial models in the aviation sector. This method can be linked to CFD analyses to dynamically reproduce the ice growth. Findings To verify the consistency of the proposed approach, one of the most challenging ice profile selected in the LEWICE manual was replicated and simulated through CFD. To showcase the effectiveness of this technique, predefined ice profiles were automatically applied on two-dimensional (2D) and three-dimensional (3D) cases using both commercial and open-source CFD solvers. Practical implications If ice accreted shapes are available, the meshless characteristic of the proposed approach enables its coupling with the CFD solvers currently supported by the RBF4AERO platform including OpenFOAM, SU2 and ANSYS Fluent. The advantages provided by the use of RBF are the high performance and reliability, due to the fast application of mesh smoothing and the accuracy in controlling surface mesh nodes. Originality/value As far as authors’ knowledge is concerned, this is the first time in scientific literature that RBF are proposed to handle icing simulations. Due to the meshless characteristic of the RBF mesh morphing, the proposed approach is cross solver and can be used for both 2D and 3D geometries.

Hospitality situations, consumer expertise, and perceptions of wine attributes: three empirical studies

2019 ◽  
Vol 31 (1) ◽  
pp. 68-88 ◽  
Author(s):  
Dale F. Duhan ◽  
Shannon B. Rinaldo ◽  
Natalia Velikova ◽  
Tim Dodd ◽  
Brent Trela
Keyword(s):  
Empirical Studies ◽  
Three Dimensional ◽  
Total Sample ◽  
Dimensional Structure ◽  
Product Knowledge ◽  
Product Attributes ◽  
Content Type ◽  
Wine Consumption ◽  
Consumer Expertise ◽  
Academic Researchers

PurposeWine choices are not always fully understood by academic researchers or the industry. This paper aims to outline and test a theoretical model proposing that wine consumption may be dependent on differences in consumer expertise, the hospitality situation, characteristics of the wine itself and an interaction of these variables.Design/methodology/approachThree empirical studies (total sample size = 356) tested these theoretical propositions. Consumers with varying levels of wine knowledge were presented with experimental vignettes showing videos of wine opening and pouring and were asked to pair wines with hospitality situations.FindingsStudy 1 found that consumers with low product knowledge were more sensitive to hospitality situations and extrinsic product attributes (closures) than were the experts. Study 2 found that wine hospitality situations fall into three predicted categories, namely, food, friends and formality, although contrary to prediction, the presence of food was the weakest predictors. Study 3 demonstrated the robustness of the three-dimensional structure of wine hospitality situations.Practical implicationsThese studies provided important practical information because targeting various market segments requires the industry to know what product attributes are favored by different groups of consumers different situations.Originality/valuePrevious researchers have discussed the difficulty of measuring consumption situations. By limiting these studies to wine consumption within hospitality situations, the authors learned much about how consumers’ characteristics, product attributes and the situations interact to influence not only product assessments but also choices.

scholarly journals Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher E. Morgan ◽  
Wei Huang ◽  
Susan D. Rudin ◽  
Derek J. Taylor ◽  
James E. Kirby ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Bound States ◽  
Antimicrobial Agents ◽  
Gram Negative ◽  
Content Type ◽  
Antibiotic Development ◽  
Cryo Electron Microscopy ◽  
70S Ribosome

ABSTRACT Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections. IMPORTANCE Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

scholarly journals HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

2015 ◽  
Vol 82 (2) ◽  
pp. 724-731 ◽  
Author(s):  
Hong-Jun Chao ◽  
Yan-Fei Chen ◽  
Ti Fang ◽  
Ying Xu ◽  
Wei E. Huang ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Environmental Protection Agency ◽  
High Performance ◽  
Specific Activity ◽  
Gene Knockout ◽  
Transcriptional Analysis ◽  
Ring Cleavage ◽  
Protection Agency ◽  
Content Type ◽  
Priority Pollutant

ABSTRACTIn addition to growing onp-cresol,Pseudomonas putidaNCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of thepara-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the genehipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg−1and showed apparentKmvalues of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similarKmvalues for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated thathipHis essential for 2,4-xylenol catabolism but not forp-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.

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