scholarly journals Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of Bacillus anthracis by Binding to the Segment of PA between Residues 686 and 694

2005 ◽  
Vol 49 (8) ◽  
pp. 3414-3420 ◽  
Author(s):  
Emmanuelle Laffly ◽  
Ludivine Danjou ◽  
Florence Condemine ◽  
Dominique Vidal ◽  
Emmanuel Drouet ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Germ Line ◽  
Cell Receptor ◽  
High Affinity ◽  
Genes Encoding ◽  
Anthrax Infection ◽  
Equilibrium Dissociation ◽  
High Degree ◽  
Encoding Genes ◽  
Selection Of

ABSTRACT Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA83). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA83 to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (107 clones). We isolated a high-affinity (equilibrium dissociation constant [KD ], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 ± 0.13 nM) Fab (designated 35PA83) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA83 and is part of the region which directly interacts with the cell receptor. 35PA83 may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA83 were similar to those of a human immunoglobulin germ line and were assigned to subgroups of human V, (D), or J genes by IMGT/V-QUEST analysis. The 35PA83 framework regions were 92% identical to a representative allele of each subgroup. When compared to framework regions coded by related human germ line genes, only 2 of 74 (VH) or 75 (VK) analyzed amino acids of 35PA83 have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA83 well suited for expression as a whole primatized immunoglobulin G and demonstrates the practicality of using macaque Fabs when immunized human plasma cell donors are not available.

Selecting highly affine and well-expressed TCRs for gene therapy of melanoma

Blood ◽  
2007 ◽  
Vol 110 (10) ◽  
pp. 3564-3572 ◽  
Author(s):  
Annelies Jorritsma ◽  
Raquel Gomez-Eerland ◽  
Maarten Dokter ◽  
Willeke van de Kasteele ◽  
Yvonne M. Zoet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Phase 1 ◽  
Human Leukocyte ◽  
Tumor Infiltrating Lymphocytes ◽  
Cell Receptor ◽  
Surface Expression ◽  
High Affinity ◽  
Tcr Gene Therapy ◽  
Selection Of

Abstract A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

scholarly journals Selection of nitrite-degrading and biogenic amine-degrading strains and its involved genes

2020 ◽  
Vol 4 (4) ◽  
pp. 225-235
Author(s):  
Yuxin Li ◽  
Zhihui Yu ◽  
Yingchun Zhu ◽  
Zhixiang Cao
Keyword(s):  
High Performance ◽  
Histidine Decarboxylase ◽  
Meat Products ◽  
Starter Cultures ◽  
Health Concern ◽  
Fermented Sausages ◽  
Degradation Rates ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Selection Of

Abstract Objectives Accumulation of nitrite and biogenic amines (BAs) in fermented meat products is a matter of public health concern. The study aimed to screen nitrite-degrading and BA-degrading strains from sour porridges and sausages and bacon products in China. Materials and Methods After screening out 12 strains, the degradation of nitrite, the degradation of BAs, the activities of nitrite-reducing enzymes, and the detection of genes involved in the BAs were assessed by spectrophotometry method with hydrochloric acid naphthalene ethylenediamine, high-performance liquid chromatography, GENMED kit, and polymerase chain reaction, respectively. Results Pediococcus pentosaceus labelled M SZ1 2 and M GC 2, Lactobacillus plantarum labelled M SZ2 2, and Staphylococcus xylosus labelled Y CC 3 were selected. The activity of nitrite-reducing enzyme in M SZ2 2 was 2.663 units/mg. The degradation rate of total BAs of M SZ2 2 was 93.24%. The degradation rates of nitrite and BAs of M SZ1 2 were 86.49% and 37.87%, respectively. The activity of nitrite-reducing enzyme in M SZ1 2 was up to 1.962 units/mg. M GC 2 showed higher degradation rates of nitrite (89.19%) and Y CC 3 showed higher degradation rates of BAs (36.16%). The genes encoding the multicopper oxidases (suf I/D2EK17) were detected in the four strains, which also did not contain BAs (histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), ornithine decarboxylase (odc), lysine decarboxylase (ldc)) formation encoding genes. Conclusion These four strains (M SZ1 2, M GC 2, M SZ2 2, and Y CC 3) are promising candidates to use as starter cultures for nitrite and BAs in fermented sausages.

scholarly journals High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

2007 ◽  
Vol 51 (8) ◽  
pp. 2758-2764 ◽  
Author(s):  
Thibaut Pelat ◽  
Michael Hust ◽  
Emmanuelle Laffly ◽  
Florence Condemine ◽  
Chantal Bottex ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Germ Line ◽  
Lethal Factor ◽  
Antibody Fragment ◽  
Human Antibody ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Vitro Assay ◽  
High Affinity

ABSTRACT The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 × 108 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ± 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

A Study of Different Methodologies Helpful in the Identification of Offline Handwritten Script

2018 ◽  
Vol 6 (6) ◽  
pp. 307
Author(s):  
Manish M. Kayasth ◽  
Bharat C. Patel
Keyword(s):  
Feature Extraction ◽  
Character Recognition ◽  
Recognition Rate ◽  
Recognition System ◽  
Post Processing ◽  
Classification Technique ◽  
Scanned Image ◽  
Gujarati Language ◽  
High Degree ◽  
Selection Of

The entire character recognition system is logically characterized into different sections like Scanning, Pre-processing, Classification, Processing, and Post-processing. In the targeted system, the scanned image is first passed through pre-processing modules then feature extraction, classification in order to achieve a high recognition rate. This paper describes mainly on Feature extraction and Classification technique. These are the methodologies which play an important role to identify offline handwritten characters specifically in Gujarati language. Feature extraction provides methods with the help of which characters can identify uniquely and with high degree of accuracy. Feature extraction helps to find the shape contained in the pattern. Several techniques are available for feature extraction and classification, however the selection of an appropriate technique based on its input decides the degree of accuracy of recognition. 

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

scholarly journals Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

2021 ◽  
Author(s):  
Fatma Ben Abid ◽  
Clement K. M. Tsui ◽  
Yohei Doi ◽  
Anand Deshmukh ◽  
Christi L. McElheny ◽  
...  
Keyword(s):  
Common Species ◽  
Clinical Samples ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Sequence Types ◽  
Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

Development of a Performance-Related Framework for Asphalt Mixture Design for the Illinois Tollway

2021 ◽  
pp. 036119812110148
Author(s):  
Behnam Jahangiri ◽  
Punyaslok Rath ◽  
Hamed Majidifard ◽  
William G. Buttlar
Keyword(s):  
Asphalt Mixture ◽  
Field Performance ◽  
Compact Tension ◽  
T Test ◽  
Novel Approach ◽  
Final Adjustment ◽  
Development And Validation ◽  
High Degree ◽  
Selection Of

Various agencies have begun to research and introduce performance-related specifications (PRS) for the design of modern asphalt paving mixtures. The focus of most recent studies has been directed toward simplified cracking test development and evaluation. In some cases, development and validation of PRS has been performed, building on these new tests, often by comparison of test values to accelerated pavement test studies and/or to limited field data. This study describes the findings of a comprehensive research project conducted at Illinois Tollway, leading to a PRS for the design of mainline and shoulder asphalt mixtures. A novel approach was developed, involving the systematic establishment of specification requirements based on: 1) selection of baseline values based on minimally acceptable field performance thresholds; 2) elevation of thresholds to account for differences between short-term lab aging and expected long-term field aging; 3) further elevation of thresholds to account for variability in lab testing, plus variability in the testing of field cores; and 4) final adjustment and rounding of thresholds based on a consensus process. After a thorough evaluation of different candidate cracking tests in the course of the project, the Disk-shaped Compact Tension—DC(T)—test was chosen to be retained in the Illinois Tollway PRS and to be presented in this study for the design of crack-resistant mixtures. The DC(T) test was selected because of its high degree of correlation with field results and its excellent repeatability. Tailored Hamburg rut depth and stripping inflection point thresholds were also established for mainline and shoulder mixes.

Precise selection of aptamers targeting PD-L1 positive small extracellular vesicles on magnetic chips

2021 ◽  
Vol 57 (29) ◽  
pp. 3555-3558
Author(s):  
Hao-Yu Dong ◽  
Qi-Hui Xie ◽  
Dai-Wen Pang ◽  
Gang Chen ◽  
Zhi-Ling Zhang
Keyword(s):  
Extracellular Vesicles ◽  
High Affinity ◽  
Selection Of

High affinity aptamers that target small extracellular vesicles displaying PD-L1 in its natural conformation were successfully selected.

scholarly journals Molecular investigation of an outbreak associated with total parenteral nutrition contaminated with NDM-producing Leclercia adecarboxylata

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Elvira Garza-González ◽  
Paola Bocanegra-Ibarias ◽  
Eduardo Rodríguez-Noriega ◽  
Esteban González-Díaz ◽  
Jesús Silva-Sanchez ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Carbapenem Resistance ◽  
Clonal Diversity ◽  
Molecular Characteristics ◽  
Molecular Evidence ◽  
Carbapenem Resistant ◽  
Parental Nutrition ◽  
Genes Encoding ◽  
Main Lineage ◽  
Encoding Genes

Abstract Background This study aimed to determine the epidemiological, microbiological, and molecular characteristics of an outbreak of carbapenem-resistant Leclercia adecarboxylata in three hospitals associated with the unintended use of contaminated total parental nutrition (TPN). Methods For 10 days, 25 patients who received intravenous TPN from the same batch of a formula developed sepsis and had blood cultures positive for L. adecarboxylata. Antimicrobial susceptibility and carbapenemase production were performed in 31 isolates, including one from an unopened bottle of TPN. Carbapenemase-encoding genes, extended-spectrum β-lactamase–encoding genes were screened by PCR, and plasmid profiles were determined. Horizontal transfer of carbapenem resistance was performed by solid mating. Clonal diversity was performed by pulsed-field gel electrophoresis. The resistome was explored by whole-genome sequencing on two selected strains, and comparative genomics was performed using Roary. Results All 31 isolates were resistant to aztreonam, cephalosporins, carbapenems, trimethoprim/sulfamethoxazole, and susceptible to gentamicin, tetracycline, and colistin. Lower susceptibility to levofloxacin (51.6%) and ciprofloxacin (22.6%) was observed. All the isolates were carbapenemase producers and positive for blaNDM-1, blaTEM-1B, and blaSHV-12 genes. One main lineage was detected (clone A, 83.9%; A1, 12.9%; A2, 3.2%). The blaNDM-1 gene is embedded in a Tn125-like element. Genome analysis showed genes encoding resistance for aminoglycosides, quinolones, trimethoprim, colistin, phenicols, and sulphonamides and the presence of IncFII (Yp), IncHI2, and IncHI2A incompatibility groups. Comparative genomics showed a major phylogenetic relationship among L. adecarboxylata I1 and USDA-ARS-USMARC-60222 genomes, followed by our two selected strains. Conclusion We present epidemiological, microbiological, and molecular evidence of an outbreak of carbapenem-resistant L. adecarboxylata in three hospitals in western Mexico associated with the use of contaminated TPN.

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