scholarly journals Discovery of Antimicrobials By Mex : Massively Parallelized Growth Assays

2021 ◽  
Author(s):  
Philipp Koch ◽  
Steven Schmitt ◽  
Mathias Cardner ◽  
Niko Beerenwinkel ◽  
Sven Panke ◽  
...  
Keyword(s):  
High Throughput ◽  
Inhibitory Concentration ◽  
Growth Curves ◽  
E Coli ◽  
Antimicrobial Substances ◽  
The Past ◽  
Naturally Occurring ◽  
Functionally Diverse ◽  
Kinetics Of ◽  
Generation Sequencing

Abstract The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances. Here we present Mex, a method for the high-throughput discovery of novel antimicrobials, that relies on E.coli self-screening to determine the bioactivity of more than ten thousand naturally occurring peptides. Analysis of thousands of E. coli growth curves using next-generation sequencing enables the identification of more than 1,000 previously unknown antimicrobial peptides. Additionally, by incorporating the kinetics of growth inhibition, a first indication of the mode of action is obtained, which has implications for the ultimate usefulness of the peptides in question. The most promising peptides of the screen are chemically synthesized and their activity is determined in standardized susceptibility assays. Ten out of 15 investigated peptides efficiently eradicate bacteria at a minimal inhibitory concentration in the lower µm or upper nm range. This work represents a step-change in the high-throughput discovery of functionally diverse antimicrobials.

scholarly journals Escherichia coli Survival in, and Release from, White-Tailed Deer Feces

2014 ◽  
Vol 81 (3) ◽  
pp. 1168-1176 ◽  
Author(s):  
Andrey K. Guber ◽  
Jessica Fry ◽  
Rebecca L. Ives ◽  
Joan B. Rose
Keyword(s):  
Escherichia Coli ◽  
Fate And Transport ◽  
Exponential Model ◽  
Temperature Correction ◽  
Content Type ◽  
E Coli ◽  
Naturally Occurring ◽  
Different Temperatures ◽  
Kinetics Of ◽  
Source Materials

ABSTRACTWhite-tailed deer are an important reservoir for pathogens that can contribute a large portion of microbial pollution in fragmented agricultural and forest landscapes. The scarcity of experimental data on survival of microorganisms in and release from deer feces makes prediction of their fate and transport less reliable and development of efficient strategies for environment protection more difficult. The goal of this study was to estimate parameters for modelingEscherichia colisurvival in and release from deer (Odocoileus virginianus) feces. Our objectives were as follows: (i) to measure survival ofE. coliin deer pellets at different temperatures, (ii) to measure kinetics ofE. colirelease from deer pellets at different rainfall intensities, and (iii) to estimate parameters of models describing survival and release of microorganisms from deer feces. Laboratory experiments were conducted to studyE. colisurvival in deer pellets at three temperatures and to estimate parameters of Chick's exponential model with temperature correction based on the Arrhenius equation. Kinetics ofE. colirelease from deer pellets were measured at two rainfall intensities and used to derive the parameters of Bradford-Schijven model of bacterial release. The results showed that parameters of the survival and release models obtained forE. coliin this study substantially differed from those obtained by using other source materials, e.g., feces of domestic animals and manures. This emphasizes the necessity of comprehensive studies of survival of naturally occurring populations of microorganisms in and release from wildlife animal feces in order to achieve better predictions of microbial fate and transport in fragmented agricultural and forest landscapes.

scholarly journals Activated carbon from termite feces impregnated with silver: Preparation and evaluation of antimicrobial activity and ecotoxicity

2020 ◽  
Vol 17 (2) ◽  
pp. 1-24
Author(s):  
Giovanna Manesco ◽  
Carla Albertina Demarchi ◽  
Alexandre Bella Cruz ◽  
Jean Felipe Fossá Calisto ◽  
Rafael Martello ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Microbial Growth ◽  
Inhibitory Concentration ◽  
Chlorophyll Synthesis ◽  
Artemia Salina ◽  
Water Disinfection ◽  
E Coli ◽  
Seedling Germination ◽  
Kinetics Of ◽  
Preparation And Evaluation

Silver has a potent antimicrobial action, but its use is still restricted due to toxicity and aggregation problems. Impregnation with active carbon (AC) has been suggested as an alternative to solve this problem. In this study, termite feces were used to prepare composites containing silver nanoparticles and AC. Reducing agents and different amounts of H2 SO4 and silver were used. Antimicrobial activity was assessed using the minimum inhibitory concentration (MIC) and the kinetics of microbial growth of S. aureus, E. coli and C. albicans. Phytotoxicity and acute toxicity were assessed for Cucumis sativus and Artemia salina. The composites that presented MIC 500 μg/mL for S. aureus and E. coli were AC25Ag (6 μg/mL Ag) and AC25AgB (78 μg/mL Ag), respectively. AC6AgS showed antimicrobial activity in C. albicans with MIC of 500 μg/mL (23 μg/mL Ag). AC25AgB inhibited the growth of S. aureus and E. coli for 48 and 32 h, respectively. At a concentration of 10 g/L, the compounds showed deleterious effects on seedling germination and elongation and on the chlorophyll synthesis of C. sativus. The composites showed no toxicity to A. salina. Based on these results, AC25AgB has the potential to be used in water disinfection.

scholarly journals Capillary electrophoresis-based functional genomics screening to discover novel archaeal DNA modifying enzymes

2021 ◽  
Author(s):  
Kelly M. Zatopek ◽  
Samantha L. Fossa ◽  
Katharina Bilotti ◽  
Paul J. Caffrey ◽  
Léa Chuzel ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Functional Genomics ◽  
High Throughput ◽  
Endonuclease Activity ◽  
Dna Endonuclease ◽  
E Coli ◽  
Ap Lyase ◽  
Generation Sequencing

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis ( T. kodakarensis ). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli . RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

ABCs of genomics

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 316-323 ◽  
Author(s):  
Stefan K. Bohlander
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Analysis ◽  
High Throughput ◽  
Patient Management ◽  
Genetic Basis ◽  
Day Treatment ◽  
Malignant Diseases ◽  
The Past ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Our genome, the 6 billion bp of DNA that contain the blueprint of a human being, has become the focus of intense interest in medicine in the past two decades. Two developments have contributed to this situation: (1) the genetic basis of more and more diseases has been discovered, especially of malignant diseases, and (2) at the same time, our abilities to analyze our genome have increased exponentially through technological breakthroughs. We can expect genomics to become ever more relevant for day-to-day treatment decisions and patient management. It is therefore of great importance for physicians, especially those who are treating patients with malignant diseases, to become familiar with our genome and the technologies that are currently available for genomics analysis. This review provides a brief overview of the organization of our genome, high-throughput sequence analysis methods, and the analysis of leukemia genomes using next-generation sequencing (NGS) technologies.

scholarly journals Rapid, direct detection of bacterial Topoisomerase 1-DNA adducts by RADAR/ELISA

2020 ◽  
Author(s):  
Devapriya Sinha ◽  
Kostantin Kiianitsa ◽  
David R. Sherman ◽  
Nancy Maizels
Keyword(s):  
Dna Adducts ◽  
High Throughput ◽  
Drug Targets ◽  
Direct Detection ◽  
E Coli ◽  
Topoisomerase 1 ◽  
Cscl Density Gradient ◽  
Chaotropic Salts ◽  
Gradient Fractionation ◽  
Kinetics Of

AbstractTopoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by “RADAR fractionation”, a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

scholarly journals Teeth Whitening and Antibacterial Effects of Juglans regia Bark: A Preliminary Study

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Riham Al-Rawi ◽  
Yusra Bashir ◽  
Aseel Mustafa ◽  
Mennatalla Omar ◽  
Noor AL-Rawi ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Juglans Regia ◽  
Ethanol Extract ◽  
Dilution Method ◽  
Antimicrobial Effects ◽  
E Coli ◽  
The Past ◽  
Serial Dilution Method ◽  
Preliminary Study

Objectives. Natural folk medicines with antimicrobial effects have been under investigation during the past decades. The aim of this study was to evaluate the teeth whitening and antimicrobial effects of ethanol extract of Persian walnut “Juglans regia” barks. Materials and Methods. Minimum inhibitory concentration (MIC) was determined using a broth microdilution assay which was conducted through a 2-fold serial dilution method, and a whitening experiment was done in vitro on extracted teeth, with a pH test being performed on 2-fold dilutions of the ethanol extract. Result. It was found that the MIC for Enterobacter and E. coli and Staphylococcus and Pseudomonas was found to be 5 mg/ml and 2.5 mg/ml, respectively. Both dilutions were found to be acidic, and the extract of Juglans regia bark also demonstrated the ability of teeth whitening. Conclusion. This study supports the use of Juglans regia bark as a natural product in dentistry because of the confirmed antimicrobial ability as well as its whitening effect. Clinical Relevance. Herb extract might be incorporated within commercially available kinds of toothpaste to enhance its whitening and antimicrobial effects.

scholarly journals A high-resolution map of bacteriophage øX174 transcription

2020 ◽  
Author(s):  
Dominic Y. Logel ◽  
Paul R. Jaschke
Keyword(s):  
High Resolution ◽  
Structural Biology ◽  
Transcriptional Activity ◽  
Antisense Transcription ◽  
Sigma Factors ◽  
E Coli ◽  
The Past ◽  
High Resolution Map ◽  
Model Virus ◽  
Generation Sequencing

AbstractBacteriophage øX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiome, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from øX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical øX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae family, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced.

scholarly journals In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP+

2020 ◽  
Author(s):  
Liliana Calzadiaz Ramirez ◽  
Carla Calvó-Tusell ◽  
Gabriele M. M. Stoffel ◽  
Steffen N. Lindner ◽  
Sílvia Osuna ◽  
...  
Keyword(s):  
High Efficiency ◽  
Screening Methods ◽  
Nadph Regeneration ◽  
E Coli ◽  
Multiple Parameters ◽  
Naturally Occurring ◽  
Dynamics Simulations ◽  
Kinetics Of ◽  
Enzyme Variant

AbstractEfficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usual specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity towards NADP+, and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

RNA polymerase: Preparation and structural analysis of helical polymers

1984 ◽  
Vol 42 ◽  
pp. 152-153
Author(s):  
E. Loren Buhle ◽  
Pamela Rew ◽  
Ueli Aebi
Keyword(s):  
Structural Analysis ◽  
Rna Polymerase ◽  
Molecular Level ◽  
X Ray Diffraction ◽  
Helical Polymers ◽  
X Ray ◽  
E Coli ◽  
The Past ◽  
Ordered Arrays ◽  
Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

Stress management: Corpus-based insights into vernacular interpretations of stress

2014 ◽  
Vol 10 (1) ◽  
pp. 81-93
Author(s):  
Laurel Smith Stvan
Keyword(s):  
Traumatic Stress ◽  
American English ◽  
Physical Symptom ◽  
The Body ◽  
Post Traumatic Stress ◽  
The Past ◽  
Academic Texts ◽  
Naturally Occurring ◽  
Post Traumatic ◽  
Semantic Shift

Examination of the term stress in naturally occurring vernacular prose provides evidence of three separate senses being conflated. A corpus analysis of 818 instances of stress from non-academic texts in the Corpus of Contemporary American English (COCA) and the Corpus of American Discourses on Health (CADOH) shows a negative prosody for stress, which is portrayed variously as a source outside the body, a physical symptom within the body and an emotional state. The data show that contemporary speakers intermingle the three senses, making more difficult a discussion between doctors and patients of ways to ‘reduce stress’, when stress might be interpreted as a stressor, a symptom, or state of anxiety. This conflation of senses reinforces the impression that stress is pervasive and increasing. In addition, a semantic shift is also refining a new sense for stress, as post-traumatic stress develops as a specific subtype of emotional stress whose use has increased in circulation in the past 20 years.

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