scholarly journals Star-Polymer-DNA Gels Showing Highly Predictable and Tunable Mechanical Responses

2021 ◽  
Author(s):  
Masashi Ohira ◽  
Takuya Katashima ◽  
Mitsuru Naito ◽  
Daisuke Aoki ◽  
Yusuke Yoshikawa ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Melting Curve ◽  
Star Polymer ◽  
Melting Curve Analysis ◽  
Mechanical Stimuli ◽  
Dissociation Kinetics ◽  
Good Correspondence ◽  
Require Time ◽  
Mechanical Responses ◽  
Thermodynamic Potentials

Dynamically crosslinked gels are appealing materials for applications that require time-dependent mechanical responses. DNA duplexes are ideal crosslinkers for building such gels because of their excellent sequence addressability and flexible tunability in bond energy. However, the mechanical responses of most DNA gels are complicated and unpredictable despite the high potential of DNA. Here, we demonstrate a DNA gel with a highly homogeneous gel network and well-predictable mechanical behaviors by using a pair of star-polymer-DNA precursors with presimulated DNA sequences showing the two-state transition. The melting curve analysis of the DNA gels reveals the good correspondence between the thermodynamic potentials of the DNA crosslinkers and the presimulated values by DNA calculators. Stress-relaxation tests and dissociation kinetics measurements show that the macroscopic relaxation time of the DNA gels is approximately equal to the lifetime of the DNA crosslinkers over four orders of magnitude from 0.1-2,000 sec. Furthermore, a series of durability tests find the DNA gels are hysteresis-less and self-healable after the applications of repeated temperature and mechanical stimuli. These results demonstrate the great potential of star-polymer-DNA precursors for building gels with predictable and tunable viscoelastic properties, suitable for applications such as stress-response extracellular matrices, injectable solids, and soft robotics.

scholarly journals Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

2007 ◽  
Vol 53 (12) ◽  
pp. 2042-2050 ◽  
Author(s):  
Brent C Satterfield ◽  
David A Kulesh ◽  
David A Norwood ◽  
Leonard P Wasieloski ◽  
Michael R Caplan ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
False Positive ◽  
Yersinia Pseudotuberculosis ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Specific Cell ◽  
Single Nucleotide ◽  
Positive Results

AbstractBackground: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation.Methods: Tentacle Probes™, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan–minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve.Results: With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred.Conclusions: The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.

scholarly journals Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15) mRNA in Various Human Cell Types

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Sang Mee Hwang ◽  
Mi Jung Kim ◽  
Ho Eun Chang ◽  
Yun Ji Hong ◽  
Taek Soo Kim ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Human Platelet ◽  
Human Fibroblasts ◽  
Leukemia Cell ◽  
Embryonic Stem ◽  
Melting Curve ◽  
Leukemia Cell Line ◽  
Melting Curve Analysis ◽  
Platelet Antigen ◽  
Human Platelet Antigen

CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

scholarly journals Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

2005 ◽  
Vol 43 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Kijeong Kim ◽  
Juwon Seo ◽  
Katherine Wheeler ◽  
Chulmin Park ◽  
Daewhan Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis

scholarly journals G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

2014 ◽  
Vol 112 (2) ◽  
pp. 590-595 ◽  
Author(s):  
Timothy Connelly ◽  
Yiqun Yu ◽  
Xavier Grosmaitre ◽  
Jue Wang ◽  
Lindsey C. Santarelli ◽  
...  
Keyword(s):  
G Protein ◽  
Sensory Neurons ◽  
Ectopic Expression ◽  
Host Cells ◽  
Odorant Receptors ◽  
Olfactory Sensory Neurons ◽  
Mechanical Stimuli ◽  
Genetic Ablation ◽  
Mechanical Responses ◽  
G Protein Coupled

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction.

scholarly journals Diagnosis of α-1-Antitrypsin Deficiency: An Algorithm of Quantification, Genotyping, and Phenotyping

2006 ◽  
Vol 52 (12) ◽  
pp. 2236-2242 ◽  
Author(s):  
Melissa R Snyder ◽  
Jerry A Katzmann ◽  
Malinda L Butz ◽  
Ping Yang ◽  
D Brian Dawson ◽  
...  
Keyword(s):  
Laboratory Testing ◽  
Null Allele ◽  
Melting Curve ◽  
Curve Analysis ◽  
Laboratory Evaluation ◽  
Melting Curve Analysis ◽  
Genotype Assay ◽  
Antitrypsin Deficiency ◽  
Phenotype Analysis ◽  
A1at Deficiency

Abstract Background: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2630-2640 ◽  
Author(s):  
J. T. Tambong ◽  
R. Xu ◽  
E. S. P. Bromfield
Keyword(s):  
Root Zone ◽  
Phylogenetic Analyses ◽  
Melting Curve ◽  
Pcr Amplification ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Its1 Region ◽  
A Genome ◽  
Pure Cultures ◽  
Intercalating Dye

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNAIle and tRNAAla in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

scholarly journals Polymorphism rs368234815 of interferon lambda 4 gene and spontaneous clearance of hepatitis C virus in haemodialysis patients: a case-control study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Alicja E. Grzegorzewska ◽  
Adrianna Mostowska ◽  
Monika K. Świderska ◽  
Wojciech Marcinkowski ◽  
Ireneusz Stolarek ◽  
...  
Keyword(s):  
Survival Analysis ◽  
Cancer Susceptibility ◽  
Melting Curve ◽  
Hcv Infection ◽  
Rank Test ◽  
Melting Curve Analysis ◽  
Hcv Rna ◽  
Spontaneous Clearance ◽  
Polymorphism Analysis ◽  
Spontaneous Hcv Clearance

Abstract Background In non-uremic subjects, IFNL4 rs368234815 predicts HCV clearance. We investigated whether rs368234815 is associated with spontaneous HCV clearance in haemodialysis patients and whether it is a stronger predictor of HCV resolution than the IFNL polymorphisms already associated with HCV clearance in dialysis subjects. We also evaluated an association of rs368234815 with patients` survival and alterations in transcription factor binding sites (TFBS) caused by IFNL polymorphisms. Methods Among 161 haemodialysis patients with positive anti-HCV antibodies, 68 (42.2%) spontaneously resolved HCV infection, whereas 93 remained HCV RNA positive. Patients were tested for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, IFNL4 rs368234815, and near IFNL4 rs8099917. IFNL4 rs368234815 polymorphism (TT/TT, ΔG/TT, ΔG/ΔG) was genotyped by restriction fragment length polymorphism analysis; other IFNL polymorphisms - by high resolution melting curve analysis. We used the Kaplan-Meier method with the log-rank test for survival analysis. In silico analysis included the use of ENCODE TFBS ChIP-seq data, HOCOMOCO, JASPAR CORE, and CIS-BP databases, and FIMO software. Results The probability (OR, 95%CI, P) of spontaneous HCV clearance for rs368234815 TT/TT patients was higher than for the ΔG allele carriers (2.63, 1.38–5.04, 0.003). This probability for other major homozygotes varied between 2.80, 1.45–5.43, 0.002 for rs12980275 and 2.44, 1.27–4.69, 0.007 for rs12979860. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance (6.38, 1.69–24.2, 0.003). Survival analysis suggested an association of the ΔG allele with mortality due to neoplasms (log-rank P = 0.005). The rs368234815 ∆G allele caused TFBS removal for PLAGL1. Conclusions In haemodialysis patients, the association of rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms only in the additive mode of inheritance. However, identifying the homozygosity in the variant ∆G allele of rs368234815 means a more potent prediction of persistent HCV infection in haemodialysis subjects that we observe in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms. Removal of PLAGL1 TFBS in subjects harbouring the rs368234815 ∆G allele may contribute to cancer susceptibility. The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed subjects.

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