Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.

OBJECTIVE: The aim of this study is to assess the microbial contamination of three different brands of esthetic elastomeric ligatures.
METHODS: Different brands of esthetic ligatures (Unistick Pearl [American Orthodontics, Sheboygan, WI, USA], Power Sticks Pearl [Ortho Technology, Tampa, FL, USA], and Ease [Obscure, 3M Unitek, Monrovia, CA, USA]) were randomly assigned to permanent canines of 25 patients (aged 11-18 years) undergoing corrective orthodontic treatment. After 30 days, the ligatures were removed, processed, and the biofilm composition was analyzed by checkerboard DNA-DNA hybridization for 40 bacterial species. The microbiological data were analyzed using a nonparametric mixed model.
RESULTS: The ligatures presented intense microbial contamination after 30 days, but no statistically significant differences were observed among the three groups (p > 0.05). The levels of the evaluated individual species and proportions of the microbial complexes showed no statistically significant differences among the ligature groups (p > 0.05).
CONCLUSIONS: Esthetic elastomeric ligatures became multicolonized by several bacterial species after 30 days of exposure to the oral cavity. However, no relevant differences were observed among the biofilm composition formed on the different ligature brands.
UNASSIGNED: ZIELSETZUNG: Ziel dieser Studie ist es, die Keimbelastung von 3 verschiedenen Fabrikaten ästhetischer Elastomer-Ligaturen zu untersuchen.
UNASSIGNED: Ästhetische Ligaturen verschiedener Hersteller (Unistick Pearl [American Orthodontics, Sheboygan, WI, USA], Power Sticks Pearl [Ortho Technology, Tampa, FL, USA] und Ease [Obscure, 3M Unitek, Monrovia, CA, USA]) wurden nach dem Zufallsprinzip den bleibenden Eckzähnen von 25 Patienten (im Alter von 11–18 Jahren) zugeordnet, die eine korrigierende kieferorthopädische Behandlung erhielten. Nach 30 Tagen wurden die Ligaturen entfernt und aufbereitet, die Zusammensetzung des Biofilms wurde durch DNA-DNA-Hybridisierung im Schachbrettverfahren auf 40 Bakterienarten hin überprüft. Die mikrobiologischen Daten wurden mit einem nichtparametrischen gemischten Modell analysiert.
UNASSIGNED: Die Ligaturen wiesen nach 30 Tagen eine starke mikrobielle Kontamination auf, allerdings wurden keine statistisch signifikanten Unterschiede zwischen den 3 Gruppen festgestellt (p > 0,05). Die Spiegel der einzelnen ausgewerteten Spezies und die Anteile der mikrobiellen Komplexe zeigten keine statistisch signifikanten Unterschiede zwischen den Ligaturgruppen (p > 0,05).
UNASSIGNED: Ästhetische Elastomer-Ligaturen wurden nach 30-tägiger Exposition in der Mundhöhle von verschiedenen Bakterienarten mehrfach besiedelt. Es wurden jedoch keine relevanten Unterschiede in der Zusammensetzung der auf den verschiedenen Ligaturmarken gebildeten Biofilme festgestellt.

CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.

DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory\'s work on developing chemical tools to study DNA repair processes in vitro, as well as in cells and tissues, and what we have learned by applying them.We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage.We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both in vitro and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses.To investigate cellular nucleotide sanitation pathways, we designed a series of \"two-headed\" nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme.We proceed to discuss the development of a \"universal\" probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.

The ongoing COVID-19 pandemic dictated new priorities in biomedicine research. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a single-stranded positive-sense RNA virus. In this pilot study, we optimized our padlock assay to visualize genomic and subgenomic regions using formalin-fixed paraffin-embedded placental samples obtained from a confirmed case of COVID-19. SARS-CoV-2 RNA was localized in trophoblastic cells. We also checked the presence of the virion by immunolocalization of its glycoprotein spike. In addition, we imaged mitochondria of placental villi keeping in mind that the mitochondrion has been suggested as a potential residence of the SARS-CoV-2 genome. We observed a substantial overlapping of SARS-CoV-2 RNA and mitochondria in trophoblastic cells. This intriguing linkage correlated with an aberrant mitochondrial network. Overall, to the best of our knowledge, this is the first study that provides evidence of colocalization of the SARS-CoV-2 genome and mitochondria in SARS-CoV-2 infected tissue. These findings also support the notion that SARS-CoV-2 infection can reprogram mitochondrial activity in the highly specialized maternal-fetal interface.

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end\'s length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.

Electrochemical biosensors have extremely robust applications while offering ease of preparation, miniaturization, and tunability. By adjusting the arrangement and properties of immobilized probes on the sensor surface to optimize target-probe association, one can design highly sensitive and efficient sensors. In electrochemical nucleic acid biosensors, a self-assembled monolayer (SAM) is widely used as a tunable surface with inserted DNA or RNA probes to detect target sequences. The effects of inhomogeneous probe distribution across surfaces are difficult to study experimentally due to inadequate resolution. Regions of high probe density may inhibit hybridization with targets, and the magnitude of the effect may vary depending on the hybridization mechanism on a given surface. Another fundamental question concerns diffusion and hybridization of DNA taking place on surfaces and whether it speeds up or hinders molecular recognition. We used all-atom Brownian dynamics simulations to help answer these questions by simulating the hybridization process of single-stranded DNA (ssDNA) targets with a ssDNA probe on polar, nonpolar, and anionic SAMs at three different probe surface densities. Moreover, we simulated three tightly packed probe clusters by modeling clusters with different interprobe spacing on two different surfaces. Our results indicate that hybridization efficiency depends strongly on finding a balance that allows attractive forces to steer target DNA toward probes without anchoring it to the surface. Furthermore, we found that the hybridization rate becomes severely hindered when interprobe spacing is less than or equal to the target DNA length, proving the need for a careful design to both enhance target-probe association and avoid steric hindrance. We developed a general kinetic model to predict hybridization times and found that it works accurately for typical probe densities. These findings elucidate basic features of nanoscale biosensors, which can aid in rational design efforts and help explain trends in experimental hybridization rates at different probe densities.

The grafting density of probes at sensor interface plays a critical role in the performance of biochemical sensors. However, compared with macroscopic interface, the effects of probe grafting density at nanometric confinement are rarely studied due to the limitation of precise grafting density regulation and characterization at the nanoscale. Here, we investigate the effect from the grafting density of DNA probes on ionic signal for nucleic acid detection in a cylindrical nanochannel array (with diameter of 25 nm) by combing experiments and theories. We set up a theoretical model of charge distribution from close to inner wall of nanochannels at low probe grafting density to spreading in whole space at high probe grafting density. The theoretical results fit well with the experimental results. A reverse of ionic output from signal-off to signal-on occurs with increasing probe grafting density. Low probe grafting density offers a high current change ratio that is further enhanced using long-chain DNA probes or the electrolyte with a low salt concentration. This work develops an approach to enhance performance of nanochannel-based sensors and explore physicochemical properties in nanometric confines.

The results of long-term author\'s studies of the optical and complex-forming properties of more than 30 synthetic low-molecular-weight fluorophores specific for DNA are described. These studies made it possible to significantly expand the already existing database of properties of such compounds, clarify the ideas about the patterns linking the mentioned properties of fluorophores with their structure, and formulate recommendations on designing new effective DNA-specific fluorophores. The results of these studies can be used, in particular, in the development of new rapid methods for diagnosing various diseases, biotesting of probiotic and antibiotic properties of various products and wastes, etc.