DNA sensor
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MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging. This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an "on to off" SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.
- MeSH
- biosenzitivní techniky * metody MeSH
- časná detekce nádoru metody MeSH
- kovové nanočástice * chemie MeSH
- lidé MeSH
- limita detekce MeSH
- mikro RNA * analýza genetika MeSH
- nádory hrtanu * diagnóza genetika MeSH
- nanovlákna * chemie MeSH
- Ramanova spektroskopie * metody MeSH
- zlato * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-helikasy metabolismus genetika MeSH
- DNA * metabolismus chemie MeSH
- dvouřetězcové zlomy DNA * MeSH
- fosforylace MeSH
- homeostáza genetika MeSH
- lidé MeSH
- oprava DNA * MeSH
- proteinfosfatasa 2 metabolismus genetika MeSH
- R-smyčka MeSH
- RNA-helikasy metabolismus genetika MeSH
- RNA-polymerasa II * metabolismus MeSH
- RNA * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Metals are widely utilized as implant materials for bone fixtures as well as stents. Biodegradable versions of these implants are highly desirable since patients do not have to undergo a second surgery for the materials to be removed. Attractive options for such materials are zinc silver alloys since they also offer the benefit of being antibacterial. However, it is important to investigate the effect of the degradation products of such alloys on the surrounding cells, taking into account silver cytotoxicity. Here we investigated zinc alloyed with 1 % of silver (Zn1Ag) and how differently concentrated extracts (1 %-100 %) of this material impact human umbilical vein endothelial cells (HUVECs). More specifically, we focused on free radical generation and oxidative stress as well as the impact on cell viability. To determine free radical production we used diamond-based quantum sensing as well as conventional fluorescent assays. The viability was assessed by observing cell morphology and the metabolic activity via the MTT assay. We found that 1 % and 10 % extracts are well tolerated by the cells. However, at higher extract concentrations we observed severe impact on cell viability and oxidative stress. We were also able to show that quantum sensing was able to detect significant free radical generation even at the lowest tested concentrations.
- MeSH
- biokompatibilní materiály chemie farmakologie MeSH
- endoteliální buňky pupečníkové žíly (lidské) * účinky léků MeSH
- lidé MeSH
- nanodiamanty * chemie MeSH
- oxidační stres * účinky léků MeSH
- slitiny * chemie MeSH
- stříbro toxicita chemie MeSH
- testování materiálů metody MeSH
- viabilita buněk * účinky léků MeSH
- volné radikály metabolismus MeSH
- vstřebatelné implantáty škodlivé účinky MeSH
- zinek * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.
The interaction of genotoxic environmental pollutant 2-nitrofluorene (2-NF) with double-stranded DNA has been studied using a hanging mercury drop electrode (HMDE) as an electrochemical sensor. Two types of DNA damage were investigated and electrochemically detected using cyclic voltammetry and differential pulse voltammetry: (i) DNA damage caused by the direct interaction with 2-NF and (ii) DNA damage caused by short-lived radicals generated by the electrochemical reduction of 2-NF. For the study of the direct interaction, the HMDE was modified by DNA and the interaction of DNA with 2-NF was studied after their mutual interaction right at the HMDE surface, or DNA was preincubated with 2-NF in solution and, subsequently, the interaction was studied voltammetrically. Using both detection techniques, the formation of DNA-2-NF complex was observed and the mutual interaction was interpreted as an intercalation between DNA base pairs. On the basis of obtained results, we suppose that expected formation of 8-oxoguanosine leads to guanosine-cytidine base pair interruption and DNA double-strand break formation. The binding constants (K) of the DNA-2-NF complex formed in solution and on the HMDE surface (DNA/HMDE) were determined from the changes in the voltammetric peaks of the studied analyte.
- MeSH
- DNA * metabolismus MeSH
- elektrody MeSH
- fluoreny MeSH
- poškození DNA MeSH
- rtuť * MeSH
- Publikační typ
- časopisecké články MeSH
IFI16 (Interferon inducible protein 16) is a DNA sensor responsible for innate immune response stimulation and a direct viral restriction by modulating gene expression and replication. Many IFI16-DNA binding properties were described - length-dependent and sequence-independent binding, oligomerization of IFI16 upon recognition, sliding on the DNA, and preference for supercoiled DNA. However, the question of the role of IFI16-DNA binding in distinct IFI16 functions remains unclear. Here we demonstrate two modes of IFI16 binding to DNA using atomic force microscopy and electrophoretic mobility shift assays. In our study, we show that IFI16 can bind to DNA in the form of globular complexes or oligomers depending on DNA topology and molar ratios. The stability of the complexes is different in higher salt concentrations. In addition, we observed no preferential binding with the HIN-A or HIN-B domains to supercoiled DNA, revealing the importance of the whole protein for this specificity. These results provide more profound insight into IFI16-DNA interactions and may be important in answering the question of self- and non-self-DNA binding by the IFI16 protein and potentially could shed light on the role of DNA binding in distinct IFI16 functions.
Cíl: Přehledová práce poskytuje základní praktické informace o využití biosenzorů v rychlé diagnostice virových patogenů. Výsledky: Viry díky evolučním změnám v genomu přeskakují do lidské populace, u níž vyvolávají závažné epidemie. Rychlé diagnostické nástroje POCT založené na biosenzorech umožní jejich rozpoznání pro potřeby klinické diagnostiky mimo specializované laboratoře. Kombinace těchto zařízení s technikami 3D tisku, mikrofluidních systémů, nanotechnologie a elektrochemické detekce výrazně zvyšuje využitelnost biosenzorů v laboratorní medicíně. Intenzivní nanomedicínský výzkum probíhá u celé řady virů, např. HIV, ebola, chřipka a viry hepatitid. V souvislosti s celosvětovou pandemií covid-19 se v současné době vývoj nanobiosenzorů ubírá především směrem k detekci SARS-CoV-2. Závěr: Dostupná literární data naznačují, že rychlé senzory a biosenzory mají značný klinický potenciál pro využití v POCT.
Aim: The review provides basic practical information about the use of biosensors in the rapid diagnosis of viral pathogens. Results: Thanks to evolutionary changes in the genome, viruses jump into the human population, where they cause serious epidemics. Rapid POCT diagnostic tools based on biosensors will enable their use for clinical diagnosis needs outside of specialized laboratories. The combination of these devices with the techniques of 3D printing, microfluidic systems, nanotechnology and electrochemical detection significantly increases the usability of biosensors. Intensive research is carried out on a wide range of viruses, e.g. HIV, Ebola, influenza, hepatitis viruses. In connection with the global covid-19 pandemic, the development of nanobiosensors is currently focused primarily on the detection of SARS-CoV-2. Conclusion: Available literature data suggest that fast sensors and biosensors have considerable clinical potential for the use in POCT.
- MeSH
- biosenzitivní techniky * MeSH
- DNA virů analýza MeSH
- elektrochemie MeSH
- lidé MeSH
- nanomedicína MeSH
- nanotechnologie MeSH
- point of care testing MeSH
- viry * izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
76-ročná pacientka s diabetes mellitus 1. typu v trvaní 24 rokov mala dlhodobo problém s veľkou variabilitou glykémií a hypoglykémiami, preto si prechodne aplikovala inzulín 5× denne. Ako 73-ročná prekonala náhlu cievnu mozgovú príhodu. 75-ročná prekonala spontánny odchod sludge zo žlčových ciest s prechodným znovu zhoršením kompenzácie diabetes mellitus 1. typu pri tráviacich problémoch s veľkou variabilitou glykémií, kvôli ktorej začala používať glukózový senzor bez potreby kalibrácie. Pomocou glukózového senzora bez potreby kalibrácie vie okamžite zareagovať na rôzne situácie v priebehu dňa a za krátkodobej pomoci stacionárneho bicykla si vie korigovať hyperglykémie. Tiež sa vie po náhlej cievnej mozgovej príhode za pomoci 24-hodinového monitoringu chrániť pred hypoglykémiami, ktoré ju trápili frekventovane v minulosti a zvyšovali jej riziko recidívy novej cievnej mozgovej príhody, poruchy srdcového rytmu ako aj demencie.
A 76-year-old patient with type 1 diabetes mellitus lasting 24 years had a long-term problem with high glycaemic variability and hypoglycaemia, and so she temporarily used insulin 5 times a day. At the age of 73, she overcame a sudden stroke. As a 75-year-old, she overcame a spontaneous passage of bile duct sludge with a transient deterioration of diabetes control with digestive problems and high glycaemic variability, due to which she started using a calibration-free glucose sensor. With the help of a calibration-free glucose sensor, she can react immediately to various situations during the day and with the help of a stationary bicycle, she can correct hyperglycaemia. And with the help of 24-hour monitoring, even after the sudden stroke, she also manages to protect herself against hypoglycaemia, which has afflicted her frequently in the past and increased her risk of recurrence of the stroke, heart rhythm disorder, and dementia.
With the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity.
DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors.
- MeSH
- DNA virů metabolismus MeSH
- Herpesviridae * genetika metabolismus MeSH
- infekce DNA virem * MeSH
- lidé MeSH
- Polyomavirus * genetika MeSH
- přirozená imunita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
