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Molecules (Basel, Switzerland) Dec 2019A sensitive and accurate identification of specific DNA fragments (usually containing a mutation) can influence clinical decisions. Standard methods routinely used for... (Review)
Review
A sensitive and accurate identification of specific DNA fragments (usually containing a mutation) can influence clinical decisions. Standard methods routinely used for this type of detection are PCR (Polymerase Chain Reaction, and its modifications), and, less commonly, NGS (Next Generation Sequencing). However, these methods are quite complicated, requiring time-consuming, multi-stage sample preparation, and specially trained staff. Usually, it takes weeks for patients to obtain their results. Therefore, different DNA sensors are being intensively developed by many groups. One technique often used to obtain an analytical signal from DNA sensors is Raman spectroscopy. Its modification, surface-enhanced Raman spectroscopy (SERS), is especially useful for practical analytical applications due to its extra low limit of detection. SERS takes advantage of the strong increase in the efficiency of Raman signal generation caused by a local electric field enhancement near plasmonic (typically gold and silver) nanostructures. In this condensed review, we describe the most important types of SERS-based nanosensors for genetic studies and comment on their potential for becoming diagnostic tools.
Topics: Biosensing Techniques; DNA; Metal Nanoparticles; Mutation; Nucleic Acid Conformation; Spectrum Analysis, Raman
PubMed: 31817059
DOI: 10.3390/molecules24244423 -
Biochemistry and Cell Biology =... Jun 2022Fast, accessible, and high-quality deoxyribonucleic acid (DNA) is fundamental to advancement in the life sciences that will drive forward fields such as agriculture,...
Fast, accessible, and high-quality deoxyribonucleic acid (DNA) is fundamental to advancement in the life sciences that will drive forward fields such as agriculture, energy, and medicine. Despite their importance in accelerating global progress, bioscience research and biotechnologies can also be misused, endangering humans, animals, and the environment. The ability to accidentally or deliberately endow or enhance the pathogenicity of biological systems is of particular concern. Access to DNA sequences with a clear potential for dual use should be limited to responsible and identifiable groups with legitimate uses. Yet, none of the 195 countries party to the International Health Regulations have national laws that mandate this type of screening. Many DNA providers voluntarily screen orders and absorb increased costs, but this practice is not universally adopted for a variety of reasons. This article explores the incentives and regulatory structures that can bring the screening coverage of DNA orders toward 100%, which may include expedited orders for approved customers, better tools and technology for more efficient screening, funding requirements that grantees use screened DNA, and early education in biosecurity aimed at researchers and students. Ultimately, an incentive-based multistakeholder approach to DNA screening can benefit researchers, industry, and global health security.
Topics: Biological Science Disciplines; DNA; Humans; Motivation; Research Personnel
PubMed: 35290750
DOI: 10.1139/bcb-2021-0504 -
Biochemistry Apr 2022RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and...
RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of -associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, RcoM-1 requires all three repeat sites to cooperatively bind DNA with a [] of 250 ± 10 nM and an average Hill coefficient, , of 1.7 ± 0.1. The paralog RcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.
Topics: Bacterial Proteins; Carbon Monoxide; DNA; Heme; Hemeproteins; Protein Binding; Transcription Factors
PubMed: 35394749
DOI: 10.1021/acs.biochem.2c00086 -
Cancer Reports (Hoboken, N.J.) Jan 2023With the progression of next-generation sequencing technologies, researchers have identified numerous variants of the regulator of telomere elongation helicase 1 (RTEL1)... (Review)
Review
BACKGROUND
With the progression of next-generation sequencing technologies, researchers have identified numerous variants of the regulator of telomere elongation helicase 1 (RTEL1) gene that are associated with a broad spectrum of phenotypic manifestations, including malignancies. At the molecular level, RTEL1 is involved in the regulation of the repair, replication, and transcription of deoxyribonucleic acid (DNA) and the maintenance of telomere length. RTEL1 can act both as a promotor and inhibitor of tumorigenesis. Here, we review the potential mechanisms implicated in the malignant transformation of tissues under conditions of RTEL1 deficiency or its aberrant overexpression.
RECENT FINDINGS
A major hemostatic challenge during RTEL1 dysfunction could arise from its unbalanced activity for unwinding guanine-rich quadruplex DNA (G4-DNA) structures. In contrast, RTEL1 deficiency leads to alterations in telomeric and genome-wide DNA maintenance mechanisms, ribonucleoprotein metabolism, and the creation of an inflammatory and immune-deficient microenvironment, all promoting malignancy. Additionally, we hypothesize that functionally similar molecules could act to compensate for the deteriorated functions of RTEL1, thereby facilitating the survival of malignant cells. On the contrary, RTEL1 over-expression was directed toward G4-unwinding, by promoting replication fork progression and maintaining intact telomeres, may facilitate malignant transformation and proliferation of various pre-malignant cellular compartments.
CONCLUSIONS
Therefore, restoring the equilibrium of RTEL1 functions could serve as a therapeutic approach for preventing and treating malignancies.
Topics: Humans; DNA; Neoplasms; Telomere; Tumor Microenvironment; DNA Helicases
PubMed: 36253342
DOI: 10.1002/cnr2.1735 -
Electrophoresis Sep 2022The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips...
The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence-compatible substrates. Cyclic olefin copolymer and toner-coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis. A series of separation channel designs and centrifugation conditions that provided successful loading of sieving polymer in less than 3 min was described. Separation of a 400-base DNA sizing ladder provided calculated base resolution between 3 and 4 bases, a greater than 18-fold improvement over separations on similar substrates. Finally, the accuracy and precision capabilities of these devices were demonstrated by separating and sizing DNA fragments of 147 and 167 bases as 148.62 ± 2 and 166.48 ± 3 bases, respectively.
Topics: Centrifugation; DNA; Electrophoresis; Lab-On-A-Chip Devices; Polymers
PubMed: 35656648
DOI: 10.1002/elps.202200090 -
Molekuliarnaia Biologiia 2021The base and nucleotide excision DNA repair (BER and NER) systems are aimed at removing specific types of damaged DNA, i.e., oxidized, alkylated, or deaminated bases in... (Review)
Review
The base and nucleotide excision DNA repair (BER and NER) systems are aimed at removing specific types of damaged DNA, i.e., oxidized, alkylated, or deaminated bases in the case of BER and bulky damage caused by UV radiation or chemical carcinogens in the case of NER. In some cases, however, the repair process follows a more complex scenario, which implies that the repair pathways exchange proteins and interact with each other to form a common interactome. This review describes the BER and NER mechanisms and discusses the current data on the involvement of the NER proteins in the repair of DNA lesions caused by oxidative stress and the BER proteins in the removal of bulky DNA adducts. We also discuss the role of poly(ADP-ribose) polymerase 1 in the regulation of the BER and NER processes and their coordination in the repair of complex (cluster) lesions.
Topics: DNA; DNA Damage; DNA Repair; Nucleotides; Oxidative Stress
PubMed: 33871434
DOI: 10.31857/S0026898421020129 -
Fertility and Sterility Jul 2023To determine whether discontinuation of delta-9-tetrahydrocannabinol (THC) use mitigates THC-associated changes in male reproductive health using a rhesus macaque model...
OBJECTIVE
To determine whether discontinuation of delta-9-tetrahydrocannabinol (THC) use mitigates THC-associated changes in male reproductive health using a rhesus macaque model of daily THC edible consumption.
DESIGN
Research animal study.
SETTING
Research institute environment.
PATIENT(S)
Adult male rhesus macaques (age, 8-10 years; n = 6).
INTERVENTION(S)
Chronic daily THC edible administration at medically and recreationally relevant contemporary doses followed by cessation of THC use.
MAIN OUTCOME MEASURE(S)
Testicular volume, serum male hormones, semen parameters, sperm deoxyribonucleic acid (DNA) fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing of sperm DNA.
RESULT(S)
Chronic THC use resulted in significant testicular atrophy, increased gonadotropin levels, decreased serum sex steroid levels, changes in seminal fluid proteome, and increased DNA fragmentation with partial recovery after discontinuation of THC use. For every increase of 1 mg/7 kg/day in THC dosing, there was a significant decrease in the total testicular volume bilaterally by 12.6 cm (95% confidence interval [CI], 10.6-14.5), resulting in a 59% decrease in volume. With THC abstinence, the total testicular volume increased to 73% of its original volume. Similarly, with THC exposure, there were significant decreases in the mean total testosterone and estradiol levels and a significant increase in the follicle-stimulating hormone level. With increasing THC dose, there was a significant decrease in the liquid semen ejaculate volume and weight of coagulum; however, no other significant changes in the other semen parameters were noted. After discontinuing THC use, there was a significant increase in the total serum testosterone level by 1.3 ng/mL (95% CI, 0.1-2.4) and estradiol level by 2.9 pg/mL (95% CI, 0.4-5.4), and the follicle-stimulating hormone level significantly decreased by 0.06 ng/mL (95% CI, 0.01-0.11). Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. Whole genome bisulfite sequencing identified 23,558 CpGs differentially methylated in heavy-THC vs. pre-THC sperm, with partial restoration of methylation after discontinuation of THC use. Genes associated with altered differentially methylated regions were enriched for those involved in the development and function of the nervous system.
CONCLUSION(S)
This is the first study demonstrating that discontinuation of chronic THC use in rhesus macaques partially restores adverse impacts to male reproductive health, THC-associated sperm differentially methylated regions in genes important for development, and expression of proteins important for male fertility.
Topics: Animals; Male; Macaca mulatta; Semen; Dronabinol; Epigenome; Proteome; Spermatozoa; Testosterone; Follicle Stimulating Hormone; Fertility; Estradiol; DNA; Sperm Count
PubMed: 36990913
DOI: 10.1016/j.fertnstert.2023.02.034 -
Nucleic Acids Research Sep 2020Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is...
Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.
Topics: DNA; Mass Spectrometry; Photometry; Single Molecule Imaging
PubMed: 32756898
DOI: 10.1093/nar/gkaa632 -
Biosensors Mar 2023Even today, most biomarker testing is executed in centralized, dedicated laboratories using bulky instruments, automated analyzers, and increased analysis time and... (Review)
Review
Even today, most biomarker testing is executed in centralized, dedicated laboratories using bulky instruments, automated analyzers, and increased analysis time and expenses. The development of miniaturized, faster, low-cost microdevices is immensely anticipated for substituting for these conventional laboratory-oriented assays and transferring diagnostic results directly onto the patient's smartphone using a cloud server. Pioneering biosensor-based approaches might make it possible to test biomarkers with reliability in a decentralized setting, but there are still a number of issues and restrictions that must be resolved before the development and use of several biosensors for the proper understanding of the measured biomarkers of numerous bioanalytes such as DNA, RNA, urine, and blood. One of the most promising processes to address some of the issues relating to the growing demand for susceptible, quick, and affordable analysis techniques in medical diagnostics is the creation of biosensors. This article critically discusses a short review of biosensors used for detecting nucleic acid biomarkers, and their use in biomedical prognostics will be addressed while considering several essential characteristics.
Topics: Humans; Biomarkers; Biosensing Techniques; DNA; Nucleic Acids; Reproducibility of Results
PubMed: 36979624
DOI: 10.3390/bios13030412 -
PloS One 2021Clinical metagenomics is a powerful diagnostic tool, as it offers an open view into all DNA in a patient's sample. This allows the detection of pathogens that would slip...
Clinical metagenomics is a powerful diagnostic tool, as it offers an open view into all DNA in a patient's sample. This allows the detection of pathogens that would slip through the cracks of classical specific assays. However, due to this unspecific nature of metagenomic sequencing, a huge amount of unspecific data is generated during the sequencing itself and the diagnosis only takes place at the data analysis stage where relevant sequences are filtered out. Typically, this is done by comparison to reference databases. While this approach has been optimized over the past years and works well to detect pathogens that are represented in the used databases, a common challenge in analysing a metagenomic patient sample arises when no pathogen sequences are found: How to determine whether truly no evidence of a pathogen is present in the data or whether the pathogen's genome is simply absent from the database and the sequences in the dataset could thus not be classified? Here, we present a novel approach to this problem of detecting novel pathogens in metagenomic datasets by classifying the (segments of) proteins encoded by the sequences in the datasets. We train a neural network on the sequences of coding sequences, labeled by taxonomic domain, and use this neural network to predict the taxonomic classification of sequences that can not be classified by comparison to a reference database, thus facilitating the detection of potential novel pathogens.
Topics: Algorithms; Animals; Bacteria; DNA; DNA, Bacterial; DNA, Viral; High-Throughput Nucleotide Sequencing; Humans; Metagenome; Metagenomics; Neural Networks, Computer; Viruses
PubMed: 34936673
DOI: 10.1371/journal.pone.0261548