scholarly journals A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Monica Wolfe ◽  
Sean Webb ◽  
Yaroslav Chushak ◽  
Rachel Krabacher ◽  
Yi Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
Diagnostic Tests ◽  
Limit Of Detection ◽  
Microarray Platform ◽  
Rapid Identification ◽  
Peptide Library ◽  
S Protein ◽  
Microarray Screening ◽  
Selection Of

AbstractRapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

DNA-Directed Patterning for Versatile Validation and Characterization of a Lipid-Based Nanoparticle Model of SARS-CoV-2

2021 ◽  
Author(s):  
Molly Kozminsky ◽  
Thomas Carey ◽  
Lydia L. Sohn
Keyword(s):  
High Throughput ◽  
Glass Substrate ◽  
Rapid Development ◽  
Neutralizing Antibody ◽  
S Protein ◽  
Biological Relevance ◽  
The Face ◽  
Great Control ◽  
Tetraspanin Cd63

Lipid-based nanoparticles have risen to the forefront of the COVID-19 pandemic—from encapsulation of vaccine components to modeling the virus, itself. Their rapid development in the face of the volatile nature of the pandemic requires high-throughput, highly flexible methods for characterization. DNA-directed patterning is a versatile method to immobilize and segregate lipid-based nanoparticles for subsequent analysis. DNA-directed patterning selectively conjugates oligonucleotides onto a glass substrate and then hybridizes them to complementary oligonucleotides tagged to the liposomes, thereby patterning them with great control and precision. The power of this method is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of SARS-CoV-2 —S-liposomes— which present the SARS-CoV-2 spike (S) protein on their surfaces. Patterning of a mixture of S-liposomes and liposomes that display the tetraspanin CD63 into discrete regions of a substrate is used to show that ACE2 specifically binds to S-liposomes. Importantly, DNA-directed patterning of S-liposomes is used to verify the performance of a commercially available neutralizing antibody against the S protein. Ultimately, the introduction of S-liposomes to ACE2-expressing cells demonstrates the biological relevance of DNA-directed patterning. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle.

scholarly journals Diagnosis and molecular typing of rabies virus in samples stored in inadequate conditions

2014 ◽  
Vol 8 (08) ◽  
pp. 1016-1021 ◽  
Author(s):  
Fernando J Beltran ◽  
Federico Gury Dohmen ◽  
Horacio Del Pietro ◽  
Daniel M Cisterna
Keyword(s):  
Rabies Virus ◽  
Molecular Typing ◽  
Diagnostic Tests ◽  
Limit Of Detection ◽  
Antibody Test ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Nucleoprotein Gene ◽  
Three Samples

Introduction: The exposure of nervous tissue samples to high temperatures affects the sensitivity of rabies virus diagnostic tests, causing degradation of the viral structure. This study evaluated reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis and molecular characterization of brain tissue samples in an advanced state of decomposition and poorly conserved viral isolates by comparing it with routine diagnostic tests. Methodology: A panel of three canine brain samples exposed to controlled decomposition for 7, 15, 30, and 120 days were evaluated using fluorescence antibody test (FAT), mouse inoculation test (MIT), and RT-PCR. In addition, 14 isolates of rabies variants, representing the largest circulation in Argentina, preserved in inadequate cooling for six to eight years were analyzed. Molecular typing of strains was performed using a 159-nucleotide region corresponding to the nucleoprotein gene. Results: The three samples analyzed were positive by RT-PCR at all the decomposition times evaluated, in contrast to results observed with FAT and MIT, which rapidly became negative. In addition, 100% of the inadequately preserved samples were characterized molecularly. The limit of detection of RT-PCR was 0.5 MICDL50/0.03 mL. Conclusion: RT-PCR can be useful for rabies diagnosis and typing of putrefying samples or rabies isolates stored in inadequate conditions.

scholarly journals Evaluation of the Droplet-Microarray Platform for High-Throughput Screening of Suspension Cells

2016 ◽  
Vol 22 (2) ◽  
pp. 163-175 ◽  
Author(s):  
Anna A. Popova ◽  
Claire Depew ◽  
Katya Manuella Permana ◽  
Alexander Trubitsyn ◽  
Ravindra Peravali ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Microarray Platform ◽  
Free Cell ◽  
Suspension Cells ◽  
Cell Content ◽  
Microtiter Plates ◽  
Microarray Screening ◽  
Screening Platform ◽  
Rare Cells

Phenotypic cell-based high-throughput screenings play a central role in drug discovery and toxicology. The main tendency in cell screenings is the increase of the throughput and decrease of reaction volume in order to accelerate the experiments, reduce the costs, and enable screenings of rare cells. Conventionally, cell-based assays are performed in microtiter plates, which exist in 96- to 1536-wells formats and cannot be further miniaturized. In addition, performing screenings of suspension cells is associated with risk of losing cell content during the staining procedures and incompatibility with high-content microscopy. Here, we evaluate the Droplet-Microarray screening platform for culturing, screening, and imaging of suspension cells. We demonstrate pipetting-free cell seeding and proliferation of cells in individual droplets of 3–80 nL in volume. We developed a methodology to perform parallel treatment, staining, and fixation of suspension cells in individual droplets. Automated imaging of live suspension cells directly in the droplets combined with algorithms for pattern recognition for image analysis is demonstrated. We evaluated the developed methodology by performing a dose–response study with antineoplastic drugs. We believe that the DMA screening platform carries great potential to be adopted for broad spectrum of screenings of suspension cells.

scholarly journals High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

2020 ◽  
Vol 117 (18) ◽  
pp. 9851-9856 ◽  
Author(s):  
William S. Sawyer ◽  
Neha Srikumar ◽  
Joseph Carver ◽  
Phillip Y. Chu ◽  
Amy Shen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Culture Media ◽  
Intact Protein ◽  
Complex Matrices ◽  
Cell Culture Media ◽  
Relative Quantitation ◽  
Order Of Magnitude ◽  
Selection Of

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire–mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire–time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.

scholarly journals BacCapSeq: a Platform for Diagnosis and Characterization of Bacterial Infections

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Orchid M. Allicock ◽  
Cheng Guo ◽  
Anne-Catrin Uhlemann ◽  
Susan Whittier ◽  
Lokendra V. Chauhan ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
High Throughput ◽  
Bacterial Infections ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Fold Increase ◽  
Virulence Determinants ◽  
Antimicrobial Resistance Genes

ABSTRACT We report a platform that increases the sensitivity of high-throughput sequencing for detection and characterization of bacteria, virulence determinants, and antimicrobial resistance (AMR) genes. The system uses a probe set comprised of 4.2 million oligonucleotides based on the Pathosystems Resource Integration Center (PATRIC) database, the Comprehensive Antibiotic Resistance Database (CARD), and the Virulence Factor Database (VFDB), representing 307 bacterial species that include all known human-pathogenic species, known antimicrobial resistance genes, and known virulence factors, respectively. The use of bacterial capture sequencing (BacCapSeq) resulted in an up to 1,000-fold increase in bacterial reads from blood samples and lowered the limit of detection by 1 to 2 orders of magnitude compared to conventional unbiased high-throughput sequencing, down to a level comparable to that of agent-specific real-time PCR with as few as 5 million total reads generated per sample. It detected not only the presence of AMR genes but also biomarkers for AMR that included both constitutive and differentially expressed transcripts. IMPORTANCE BacCapSeq is a method for differential diagnosis of bacterial infections and defining antimicrobial sensitivity profiles that has the potential to reduce morbidity and mortality, health care costs, and the inappropriate use of antibiotics that contributes to the development of antimicrobial resistance.

DNA-Directed Patterning for Versatile Validation and Characterization of a Lipid-Based Nanoparticle Model of SARS-CoV-2

2021 ◽  
Author(s):  
Molly Kozminsky ◽  
Thomas Carey ◽  
Lydia L. Sohn
Keyword(s):  
High Throughput ◽  
Glass Substrate ◽  
Rapid Development ◽  
Neutralizing Antibody ◽  
S Protein ◽  
Biological Relevance ◽  
The Face ◽  
Great Control ◽  
Tetraspanin Cd63

Lipid-based nanoparticles have risen to the forefront of the COVID-19 pandemic—from encapsulation of vaccine components to modeling the virus, itself. Their rapid development in the face of the volatile nature of the pandemic requires high-throughput, highly flexible methods for characterization. DNA-directed patterning is a versatile method to immobilize and segregate lipid-based nanoparticles for subsequent analysis. DNA-directed patterning selectively conjugates oligonucleotides onto a glass substrate and then hybridizes them to complementary oligonucleotides tagged to the liposomes, thereby patterning them with great control and precision. The power of this method is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of SARS-CoV-2 —S-liposomes— which present the SARS-CoV-2 spike (S) protein on their surfaces. Patterning of a mixture of S-liposomes and liposomes that display the tetraspanin CD63 into discrete regions of a substrate is used to show that ACE2 specifically binds to S-liposomes. Importantly, DNA-directed patterning of S-liposomes is used to verify the performance of a commercially available neutralizing antibody against the S protein. Ultimately, the introduction of S-liposomes to ACE2-expressing cells demonstrates the biological relevance of DNA-directed patterning. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle.

scholarly journals High-throughput identification of viral termini and packaging mechanisms in virome datasets using PhageTermVirome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julian R. Garneau ◽  
Véronique Legrand ◽  
Martial Marbouty ◽  
Maximilian O. Press ◽  
Dean R. Vik ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Computing Time ◽  
Rapid Identification ◽  
Viral Metagenomics ◽  
Genome Packaging ◽  
Viral Origin ◽  
Virus Identification ◽  
Viral Particles

AbstractViruses that infect bacteria (phages) are increasingly recognized for their importance in diverse ecosystems but identifying and annotating them in large-scale sequence datasets is still challenging. Although efficient scalable virus identification tools are emerging, defining the exact ends (termini) of phage genomes is still particularly difficult. The proper identification of termini is crucial, as it helps in characterizing the packaging mechanism of bacteriophages and provides information on various aspects of phage biology. Here, we introduce PhageTermVirome (PTV) as a tool for the easy and rapid high-throughput determination of phage termini and packaging mechanisms using modern large-scale metagenomics datasets. We successfully tested the PTV algorithm on a mock virome dataset and then used it on two real virome datasets to achieve the rapid identification of more than 100 phage termini and packaging mechanisms, with just a few hours of computing time. Because PTV allows the identification of free fully formed viral particles (by recognition of termini present only in encapsidated DNA), it can also complement other virus identification softwares to predict the true viral origin of contigs in viral metagenomics datasets. PTV is a novel and unique tool for high-throughput characterization of phage genomes, including phage termini identification and characterization of genome packaging mechanisms. This software should help researchers better visualize, map and study the virosphere. PTV is freely available for downloading and installation at https://gitlab.pasteur.fr/vlegrand/ptv.

scholarly journals Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput

mBio ◽  
2021 ◽  
Author(s):  
Ludimila Dias Almeida ◽  
Ali Salim Faraj Silva ◽  
Daniel Calixto Mota ◽  
Adrielle Ayumi Vasconcelos ◽  
Antônio Pedro Camargo ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Deletion ◽  
Rapid Identification ◽  
Potential Drug ◽  
Transporter Gene ◽  
Import And Export ◽  
Resistant Mutants ◽  
Chemical Groups ◽  
Deletion Library ◽  
Selection Of

Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

Applications of Transmission Electron Microscopy in the Characterization of Metal Machinability

1968 ◽  
Vol 26 ◽  
pp. 442-443 ◽  
Author(s):  
L.E. Murr ◽  
A.B. Draper
Keyword(s):  
Electron Microscopy ◽  
State Physics ◽  
Test Material ◽  
Milling Machine ◽  
Rotary Table ◽  
Solid State Physics ◽  
Materials Properties ◽  
Transmission Electron ◽  
Selection Of

The industrial characterization of the machinability of metals and alloys has always been a very arbitrarily defined property, subject to the selection of various reference or test materials; and the adoption of rather naive and misleading interpretations and standards. However, it seems reasonable to assume that with the present state of knowledge of materials properties, and the current theories of solid state physics, more basic guidelines for machinability characterization might be established on the basis of the residual machined microstructures. This approach was originally pursued by Draper; and our presentation here will simply reflect an exposition and extension of this research.The technique consists initially in the production of machined chips of a desired test material on a horizontal milling machine with the workpiece (specimen) mounted on a rotary table vice. A single cut of a specified depth is taken from the workpiece (0.25 in. wide) each at a new tool location.

Combinatorial Fabrication and High-Throughput Characterization of Thin Film Metal Oxide Libraries for Solar water Splitting

2018 ◽  
Author(s):  
Alfred Ludwig ◽  
Mona Nowak ◽  
Swati Kumari ◽  
Helge S. Stein ◽  
Ramona Gutkowski ◽  
...  
Keyword(s):  
Thin Film ◽  
Metal Oxide ◽  
Water Splitting ◽  
High Throughput ◽  
Solar Water ◽  
Solar Water Splitting
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