Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia

Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method.

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Osteogenesis Imperfecta: Molecular and Mesoscale Disease Mechanisms

ASME 2009 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2009-204530 ◽

2009 ◽

Author(s):

Alfonso Gautieri ◽

Sebastien Uzel ◽

Simone Vesentini ◽

Alberto Redaelli ◽

Markus J. Buehler

Keyword(s):

Mechanical Properties ◽

Osteogenesis Imperfecta ◽

Multiple Scales ◽

Genetic Disorder ◽

Single Point ◽

Single Point Mutation ◽

Mesoscale Simulation ◽

Disease Mechanisms ◽

Multiple Length Scales ◽

Collagenous Tissues

Osteogenesis Imperfecta (OI) is a genetic disorder in collagen characterized by mechanically weakened tendon and fragile bones that affects more than 1 in 10,000 individuals. Even though many studies have attempted to associate specific mutation types with phenotypic severity, the mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length-scales remain unknown. Here we show by a hierarchy of full atomistic and mesoscale simulation that OI mutations severely compromise the mechanical properties of collagenous tissues at multiple scales, from single molecules to collagen fibrils.

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Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v20i3.1825 ◽

2021 ◽

Vol 20 (3) ◽

Author(s):

Hairul Aini Hamzah ◽

Rahmatullah Sirat ◽

Mohammed Imad A. Mustafa Mahmud ◽

Roesnita Baharudin

Keyword(s):

Mutational Analysis ◽

Single Point ◽

Point Mutations ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Resistant Bacteria ◽

Single Point Mutation ◽

Phenotypic Resistance ◽

Drug Effectiveness ◽

E Coli

Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

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Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500501112 ◽

2015 ◽

Vol 112 (8) ◽

pp. 2551-2556 ◽

Cited By ~ 32

Author(s):

Michael A. Bemben ◽

Quynh-Anh Nguyen ◽

Tongguang Wang ◽

Yan Li ◽

Roger A. Nicoll ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Single Point ◽

Point Mutations ◽

Genetic Mutation ◽

Autism Spectrum ◽

Repetitive Behaviors ◽

Single Point Mutation ◽

Kinase C ◽

Human Neurons

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

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Monitoring of Pyrethroid Resistance Allele Frequency in the Common Bed Bug (Cimex lectularius) in the Republic of Korea

The Korean Journal of Parasitology ◽

10.3347/kjp.2020.58.1.99 ◽

2020 ◽

Vol 58 (1) ◽

pp. 99-102

Author(s):

Susie Cho ◽

Heung-Chul Kim ◽

Sung-Tae Chong ◽

Terry A. Klein ◽

Deok Ho Kwon ◽

...

Keyword(s):

Large Scale ◽

Pyrethroid Resistance ◽

Single Point ◽

Point Mutations ◽

Monitoring Program ◽

Cimex Lectularius ◽

Single Point Mutation ◽

Resistance Allele ◽

Bed Bugs ◽

Bed Bug

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (<i>Cimex lectularius</i>) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

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Point mutations in the beta-subunit of cytochrome b558 leading to X- linked chronic granulomatous disease

Blood ◽

10.1182/blood.v77.11.2482.bloodjournal77112482 ◽

1991 ◽

Vol 77 (11) ◽

pp. 2482-2487 ◽

Cited By ~ 1

Author(s):

BG Bolscher ◽

M de Boer ◽

A de Klein ◽

RS Weening ◽

D Roos

Keyword(s):

Chronic Granulomatous Disease ◽

Single Point ◽

Genetic Defect ◽

Point Mutations ◽

Heme Iron ◽

Beta Subunit ◽

Stable Complex ◽

Single Point Mutation ◽

Granulomatous Disease ◽

Cytochrome B558

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

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Sharp-featured Au@Ag core/shell nanocuboid synthesis and the label-free ultrasensitive SERS detection of protein single-point mutations

Materials Chemistry Frontiers ◽

10.1039/c8qm00185e ◽

2018 ◽

Vol 2 (9) ◽

pp. 1720-1724 ◽

Cited By ~ 3

Author(s):

Guopeng Tu ◽

Ntirikwendera Deogratias ◽

Meng Xu ◽

Xiaowei Li ◽

Jiajia Liu ◽

...

Keyword(s):

Single Point ◽

Point Mutations ◽

Core Shell ◽

Label Free ◽

Sers Detection ◽

Single Point Mutations

Sharp-featured Au@Ag core/shell nanocuboids were synthesized.

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mmCSM-AB: guiding rational antibody engineering through multiple point mutations

Nucleic Acids Research ◽

10.1093/nar/gkaa389 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W125-W131 ◽

Cited By ~ 2

Author(s):

Yoochan Myung ◽

Douglas E V Pires ◽

David B Ascher

Keyword(s):

Binding Affinity ◽

Single Point ◽

Point Mutations ◽

Affinity Maturation ◽

Antibody Engineering ◽

Multiple Point ◽

Single Point Mutation ◽

Atomic Interaction ◽

Therapeutic Class ◽

Antibody Design

Abstract While antibodies are becoming an increasingly important therapeutic class, especially in personalized medicine, their development and optimization has been largely through experimental exploration. While there have been many efforts to develop computational tools to guide rational antibody engineering, most approaches are of limited accuracy when applied to antibody design, and have largely been limited to analysing a single point mutation at a time. To overcome this gap, we have curated a dataset of 242 experimentally determined changes in binding affinity upon multiple point mutations in antibody-target complexes (89 increasing and 153 decreasing binding affinity). Here, we have shown that by using our graph-based signatures and atomic interaction information, we can accurately analyse the consequence of multi-point mutations on antigen binding affinity. Our approach outperformed other available tools across cross-validation and two independent blind tests, achieving Pearson's correlations of up to 0.95. We have implemented our new approach, mmCSM-AB, as a web-server that can help guide the process of affinity maturation in antibody design. mmCSM-AB is freely available at http://biosig.unimelb.edu.au/mmcsm_ab/.

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Mutations in Aspergillus fumigatus Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.2.577-581.2003 ◽

2003 ◽

Vol 47 (2) ◽

pp. 577-581 ◽

Cited By ~ 134

Author(s):

Paul A. Mann ◽

Raulo M. Parmegiani ◽

Shui-Qing Wei ◽

Cara A. Mendrick ◽

Xin Li ◽

...

Keyword(s):

Cytochrome P450 ◽

Aspergillus Fumigatus ◽

Single Point ◽

Point Mutations ◽

Drug Accumulation ◽

Resistant Isolate ◽

Single Amino Acid ◽

Single Point Mutation ◽

Genes Encoding ◽

Moderately Resistant

ABSTRACT To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 108 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [14C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14α-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

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Assessing Protein-Drug Resistance Due to Mutations via a Rigidity Analysis in silico Approach

10.29007/7gnf ◽

2020 ◽

Author(s):

Dylan Carpenter ◽

Tess Thackray ◽

Cecilia Kalthoff ◽

Filip Jagodzinski

Keyword(s):

Drug Resistance ◽

Amino Acid ◽

In Silico ◽

Single Point ◽

Point Mutations ◽

Single Point Mutation ◽

Protein Drug ◽

Ligand Complex ◽

Rigidity Analysis ◽

The Impact

A mutation to the amino acid sequence of a protein can cause a biomolecule to be resistant to the intended effects of a drug. Assessing the changes of a drug’s efficacy in response to mutations via mutagenesis wet-lab experiments is prohibitively time consuming for even a single point mutation, let alone for all possible mutations. Existing approaches for inferring mutation-induced drug resistance are available, but all of them reason about mutations of residues at or very near the protein-drug interface. However, there are examples of mutations far away from the region where the ligand binds, but which nonetheless render a protein resistant to the effects of the drug. We present a proof-of-concept computational pipeline that generates in silico the set of all possible single point mutations in a protein-ligand complex. We assess drug resistance using a graph theoretic rigidity analysis approach. Unlike existing methods, we are able to assess the impact of mutations far away from the protein-drug interface. We introduce several visualizations for exploring how amino acid substitutions both near and far away from where the ligand interacts with a protein target have a stabilizing or destabilizing effect on the protein-drug complex. We discuss our analytical approach in the context of experimental data from the literature about clinically known protein-drug interactions.

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Molecular and Mesoscale Mechanisms of Osteogenesis Imperfecta Disease

ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology ◽

10.1115/nemb2010-13160 ◽

2010 ◽

Author(s):

Sebastien Uzel ◽

Markus J. Buehler

Keyword(s):

Osteogenesis Imperfecta ◽

Molecular Model ◽

Genetic Disorder ◽

Single Point ◽

Collagen Fibrils ◽

Single Point Mutation ◽

Structural Protein ◽

Hierarchical Assembly ◽

Multiple Length Scales ◽

Collagenous Tissue

Collagen is a crucial structural protein material, formed through a hierarchical assembly of tropocollagen molecules, arranged in collagen fibrils that constitute the basis for larger-scale fibrils and fibers. Osteogenesis imperfecta is a genetic disorder in collagen characterized by mechanically weakened tendon, fragile bones, skeletal deformities and in severe cases prenatal death. Even though many studies have attempted to associate specific mutation types with phenotypic severity, the mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length-scales remain unknown. In this study, we report a series of systematic molecular scale based bottom-up computational experiments focused on pure collagenous tissue, carried out using atomistic-level molecular dynamics (MD), adaptive Poison-Boltzmann solver (APBS) calculations, and a mesoscale molecular model of collagen fibrils.

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