Non-radioactive Detection Methods for in Vitro Footprinting

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

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Models and methods to characterise levonorgestrel release from intradermally administered contraceptives

Drug Delivery and Translational Research ◽

10.1007/s13346-021-01091-5 ◽

2021 ◽

Author(s):

Adnan Al Dalaty ◽

Benedetta Gualeni ◽

Sion A. Coulman ◽

James C. Birchall

Keyword(s):

Ex Vivo ◽

Extraction Procedure ◽

In Vitro Release ◽

Reversed Phase ◽

Experimental Models ◽

Detection Methods ◽

Hormonal Contraceptives ◽

Limit Of Quantification ◽

Long Acting

AbstractMicroneedle (MN)-based technologies have been proposed as a means to facilitate minimally invasive sustained delivery of long-acting hormonal contraceptives into the skin. Intradermal administration is a new route of delivery for these contraceptives and therefore no established laboratory methods or experimental models are available to predict dermal drug release and pharmacokinetics from candidate MN formulations. This study evaluates an in vitro release (IVR) medium and a medium supplemented with ex vivo human skin homogenate (SH) as potential laboratory models to investigate the dermal release characteristics of one such hormonal contraceptive that is being tested for MN delivery, levonorgestrel (LNG), and provides details of an accompanying novel two-step liquid–liquid drug extraction procedure and sensitive reversed-phase HPLC–UV assay. The extraction efficiency of LNG was 91.7 ± 3.06% from IVR medium and 84.6 ± 1.6% from the medium supplemented with SH. The HPLC–UV methodology had a limit of quantification of 0.005 µg/mL and linearity between 0.005 and 25 µg/mL. Extraction and detection methods for LNG were exemplified in both models using the well-characterised, commercially available sustained-release implant (Jadelle®). Sustained LNG release from the implant was detected in both media over 28 days. This study reports for the first time the use of biologically relevant release models and a rapid, reliable and sensitive methodology to determine release characteristics of LNG from intradermally administered long-acting drug delivery systems. Graphical abstract

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Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

10.1101/2020.05.20.106617 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shanaya Shital Shah ◽

Stella Hartono ◽

Frédéric Chédin ◽

Wolf-Dietrich Heyer

Keyword(s):

Real Time ◽

Single Molecule ◽

Detection Methods ◽

Loop Formation ◽

Bisulfite Treatment ◽

Loop Detection ◽

D Loop ◽

Donor Choice ◽

The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

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Listeria monocytogenes survival in raw Atlantic salmon (Salmo salar) fillet under in vitro simulated gastrointestinal conditions by culture, qPCR and PMA-qPCR detection methods

LWT ◽

10.1016/j.lwt.2019.03.015 ◽

2019 ◽

Vol 107 ◽

pp. 132-137

Author(s):

Clarissa Barretta ◽

Silvani Verruck ◽

Bruna Marchesan Maran ◽

Leticia dos Santos Maurício ◽

Marília Miotto ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Atlantic Salmon ◽

Salmo Salar ◽

Detection Methods ◽

Atlantic Salmon Salmo Salar ◽

Gastrointestinal Conditions

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Model-based detection of putative synaptic connections from spike recordings with latency and type constraints

Journal of Neurophysiology ◽

10.1152/jn.00066.2020 ◽

2020 ◽

Vol 124 (6) ◽

pp. 1588-1604

Author(s):

Naixin Ren ◽

Shinya Ito ◽

Hadi Hafizi ◽

John M. Beggs ◽

Ian H. Stevenson

Keyword(s):

Linear Model ◽

Generalized Linear Model ◽

Large Scale ◽

Statistical Problem ◽

Detection Methods ◽

Multielectrode Array ◽

Synaptic Connections ◽

Synaptic Effects ◽

Synapse Detection

Detecting synaptic connections using large-scale extracellular spike recordings is a difficult statistical problem. Here, we develop an extension of a generalized linear model that explicitly separates fast synaptic effects and slow background fluctuations in cross-correlograms between pairs of neurons while incorporating circuit properties learned from the whole network. This model outperforms two previously developed synapse detection methods in the simulated networks and recovers plausible connections from hundreds of neurons in in vitro multielectrode array data.

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Development and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves

Phytopathology ◽

10.1094/phyto-07-19-0227-r ◽

2020 ◽

Vol 110 (3) ◽

pp. 547-555

Author(s):

Silvia Scibetta ◽

Giovanni E. Agosteo ◽

Ahmed Abdelfattah ◽

Maria G. Li Destri Nicosia ◽

Santa O. Cacciola ◽

...

Keyword(s):

Quantitative Pcr ◽

High Throughput Sequencing ◽

Current Knowledge ◽

Leaf Growth ◽

Its Region ◽

Qpcr Assay ◽

Detection Methods ◽

Effective Control ◽

Its2 Region

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods.

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Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2010.1491 ◽

2010 ◽

Vol 1 (1) ◽

pp. 3

Author(s):

Ivan B. Brukner ◽

Anne-Marie Larose ◽

Izabella Gorska-Flipot ◽

Maja Krajinovic ◽

Damian Labuda

Keyword(s):

Rational Design ◽

In Vitro Selection ◽

Clinical Performance ◽

Initial Step ◽

Detection Methods ◽

Ambient Temperatures ◽

Hybridization Probes ◽

Specificity And Sensitivity ◽

Hpv Types

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through i<em>n vitro </em>selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

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Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

Biomolecules ◽

10.3390/biom10030469 ◽

2020 ◽

Vol 10 (3) ◽

pp. 469

Author(s):

Hasier Eraña ◽

Jorge M. Charco ◽

Ezequiel González-Miranda ◽

Sandra García-Martínez ◽

Rafael López-Moreno ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Protein Misfolding ◽

Prion Diseases ◽

Immunohistochemical Analysis ◽

Body Fluids ◽

Detection Methods ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Prion Propagation

Transmissible spongiform encephalopathies or prion diseases are rapidly progressive neurodegenerative diseases, the clinical manifestation of which can resemble other promptly evolving neurological maladies. Therefore, the unequivocal ante-mortem diagnosis is highly challenging and was only possible by histopathological and immunohistochemical analysis of the brain at necropsy. Although surrogate biomarkers of neurological damage have become invaluable to complement clinical data and provide more accurate diagnostics at early stages, other neurodegenerative diseases show similar alterations hindering the differential diagnosis. To solve that, the detection of the pathognomonic biomarker of disease, PrPSc, the aberrantly folded isoform of the prion protein, could be used. However, the amounts in easily accessible tissues or body fluids at pre-clinical or early clinical stages are extremely low for the standard detection methods. The solution comes from the recent development of in vitro prion propagation techniques, such as Protein Misfolding Cyclic Amplification (PMCA) and Real Time-Quaking Induced Conversion (RT-QuIC), which have been already applied to detect minute amounts of PrPSc in different matrixes and make early diagnosis of prion diseases feasible in a near future. Herein, the most relevant tissues and body fluids in which PrPSc has been detected in animals and humans are being reviewed, especially those in which cell-free prion propagation systems have been used with diagnostic purposes.

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In vitro antifungal susceptibility of candidemia agents and detection of their biofilm production by two different methods

Microbiology Research ◽

10.4081/mr.2017.6916 ◽

2017 ◽

Vol 8 (1) ◽

Author(s):

Yasemin Oz ◽

Iman Qoraan ◽

Egemen Gokbolat

Keyword(s):

Production Rate ◽

Biofilm Formation ◽

Colorimetric Assay ◽

Formation Rate ◽

Antifungal Susceptibility ◽

Reference Method ◽

Antifungal Resistance ◽

Detection Methods ◽

Biofilm Production

Candida bloodstream infections are a significant cause of morbidity and mortality in hospitalized patients. The most important contribution of biofilm is the higher antifungal resistance than planktonic cells. We aimed to investigate the biofilm formation rate and antifungal susceptibility characteristics of our bloodstream isolates, and evaluate two different biofilm detection methods. A total of 200 bloodstream Candida isolates were included. The biofilms were formed on 96-well microtiter plates and measured by spectrophotometric percent transmittance and 2,3-bis(2- methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium- 5-carboxanilide colorimetric assay. In addition antifungal susceptibilities of these isolates were evaluated against caspofungin, anidulafungin and amphotericin B by reference method. Biofilm production rate was considerably high among our bloodstream isolates. The most important biofilm producer species was C. tropicalis; C. glabrata had the lowest biofilm production rate. The consistency rate between biofilm detection methods was 66%. Remarkable antifungal resistance was not observed among our isolates in general. In conclusion, biofilm production in Candida species is an important virulence factor, and its rate is considerably high in bloodstream isolates. At present, a standardized method has not been established to detect the biofilm formation.

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Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

Applied and Environmental Microbiology ◽

10.1128/aem.00929-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 5794-5803 ◽

Cited By ~ 9

Author(s):

Komlavi Anani Afanou ◽

Anne Straumfors ◽

Asbjørn Skogstad ◽

Ajay P. Nayak ◽

Ida Skaar ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Field Emission ◽

Polyclonal Antibodies ◽

Detection Methods ◽

Indoor Environments ◽

Content Type ◽

Freeze Dried ◽

Scanning Electron

ABSTRACTSubmicronic fungal fragments have been observed inin vitroaerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores fromAspergillus versicolorand high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed withA. versicolorfragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments ofA. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.

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