Carbon-Based Fluorescent Nano-Biosensors for the Detection of Cell-Free Circulating MicroRNAs.

Currently, non-communicable diseases (NCDs) have emerged as potential risks for humans due to adopting a sedentary lifestyle and inaccurate diagnoses. The early detection of NCDs using point-of-care technologies significantly decreases the burden and will be poised to transform clinical intervention and healthcare provision. An imbalance in the levels of circulating cell-free microRNAs (ccf-miRNA) has manifested in NCDs, which are passively released into the bloodstream or actively produced from cells, improving the efficacy of disease screening and providing enormous sensing potential. The effective sensing of ccf-miRNA continues to be a significant technical challenge, even though sophisticated equipment is needed to analyze readouts and expression patterns. Nanomaterials have come to light as a potential solution as they provide significant advantages over other widely used diagnostic techniques to measure miRNAs. Particularly, CNDs-based fluorescence nano-biosensors are of great interest. Owing to the excellent fluorescence characteristics of CNDs, developing such sensors for ccf-microRNAs has been much more accessible. Here, we have critically examined recent advancements in fluorescence-based CNDs biosensors, including tools and techniques used for manufacturing these biosensors. Green synthesis methods for scaling up high-quality, fluorescent CNDs from a natural source are discussed. The various surface modifications that help attach biomolecules to CNDs utilizing covalent conjugation techniques for multiple applications, including self-assembly, sensing, and imaging, are analyzed. The current review will be of particular interest to researchers interested in fluorescence-based biosensors, materials chemistry, nanomedicine, and related fields, as we focus on CNDs-based nano-biosensors for ccf-miRNAs detection applications in the medical field.

Cell-free circulating mitochondrial DNA: An emerging biomarker for airborne particulate matter associated with cardiovascular diseases.

The association of airborne particulate matter exposure with the deteriorating function of the cardiovascular system is fundamentally driven by the impairment of mitochondrial-nuclear crosstalk orchestrated by aberrant redox signaling. The loss of delicate balance in retrograde communication from mitochondria to the nucleus often culminates in the methylation of the newly synthesized strand of mitochondrial DNA (mtDNA) through DNA methyl transferases. In highly metabolic active tissues such as the heart, mtDNA's methylation state alteration impacts mitochondrial bioenergetics. It affects transcriptional regulatory processes involved in biogenesis, fission, and fusion, often accompanied by the integrated stress response. Previous studies have demonstrated a paradoxical role of mtDNA methylation in cardiovascular pathologies linked to air pollution. A pronounced alteration in mtDNA methylation contributes to systemic inflammation, an etiological determinant for several co-morbidities, including vascular endothelial dysfunction and myocardial injury. In the current article, we evaluate the state of evidence and examine the considerable promise of using cell-free circulating methylated mtDNA as a predictive biomarker to reduce the more significant burden of ambient air pollution on cardiovascular diseases.

Bioanalytical Applications of Graphene Quantum Dots for Circulating Cell-Free Nucleic Acids: A Review.

Graphene quantum dots (GQDs) are carbonaceous nanodots that are natural crystalline semiconductors and range from 1 to 20 nm. The broad range of applications for GQDs is based on their unique physical and chemical properties. Compared to inorganic quantum dots, GQDs possess numerous advantages, including formidable biocompatibility, low intrinsic toxicity, excellent dispensability, hydrophilicity, and surface grating, thus making them promising materials for nanophotonic applications. Owing to their unique photonic compliant properties, such as superb solubility, robust chemical inertness, large specific surface area, superabundant surface conjugation sites, superior photostability, resistance to photobleaching, and nonblinking, GQDs have emerged as a novel class of probes for the detection of biomolecules and study of their molecular interactions. Here, we present a brief overview of GQDs, their advantages over quantum dots (QDs), various synthesis procedures, and different surface conjugation chemistries for detecting cell-free circulating nucleic acids (CNAs). With the prominent rise of liquid biopsy-based approaches for real-time detection of CNAs, GQDs-based strategies might be a step toward early diagnosis, prognosis, treatment monitoring, and outcome prediction of various non-communicable diseases, including cancers.

Graphene Quantum-Dot-Based Nanophotonic Approach for Targeted Detection of Long Noncoding RNAs in Circulation.

Recent progress in the field of nanophotonics has opened up novel avenues for developing nanomaterial-based biosensing systems, which can detect various disease-specific biomarkers, including long noncoding RNAs (lncRNAs) known to circulate in biological fluids. Herein, we designed and developed a nanophotonic approach for rapid and specific capture of lncRNAs using oligonucleotide-conjugated graphene quantum-dot-nanoconjugates. The method offers accurate identification of the target lncRNAs with high selectivity, despite the presence of other molecules in the given sample. The observations also pointed toward the high feasibility and simplicity of the method in the selective determination of lncRNAs. Overall, the approach has the potential of assessing lncRNA expression as a function of disease initiation and progression.

Prenatal exposure to environmental pro-oxidants induces mitochondria-mediated epigenetic changes: a cross-sectional pilot study.

Mitochondria play a central role in maintaining cellular and metabolic homeostasis during vital development cycles of foetal growth. Optimal mitochondrial functions are important not only to sustain adequate energy production but also for regulated epigenetic programming. However, these organelles are subtle targets of environmental exposures, and any perturbance in the defined mitochondrial machinery during the developmental stage can lead to the re-programming of the foetal epigenetic landscape. As these modifications can be transferred to subsequent generations, we herein performed a cross-sectional study to have an in-depth understanding of this intricate phenomenon. The study was conducted with two arms: whereas the first group consisted of in utero pro-oxidant exposed individuals and the second group included controls. Our results showed higher levels of oxidative mtDNA damage and associated integrated stress response among the exposed individuals. These disturbances were found to be closely related to the observed discrepancies in mitochondrial biogenesis. The exposed group showed mtDNA hypermethylation and changes in allied mitochondrial functioning. Altered expression of mitomiRs and their respective target genes in the exposed group indicated the possibilities of a disturbed mitochondrial-nuclear cross talk. This was further confirmed by the modified activity of the mitochondrial stress regulators and pro-inflammatory mediators among the exposed group. Importantly, the disturbed DNMT functioning, hypermethylation of nuclear DNA, and higher degree of post-translational histone modifications established the existence of aberrant epigenetic modifications in the exposed individuals. Overall, our results demonstrate the first molecular insights of in utero pro-oxidant exposure associated changes in the mitochondrial-epigenetic axis. Although, our study might not cement an exposure-response relationship for any particular environmental pro-oxidant, but suffice to establish a dogma of mito-epigenetic reprogramming at intrauterine milieu with chronic illness, a hitherto unreported interaction.

Surface-enhanced Raman scattering biosensors for detection of oncomiRs in breast cancer.

Surface-enhanced Raman scattering (SERS) has emerged as one of the most promising platforms for various biosensing applications. These sensing systems encompass the advantages of specificity, ultra-high sensitivity, stability, low cost, repeatability, and easy-to-use methods. Moreover, their ability to offer a molecular fingerprint and identify the target analyte at low levels make SERS a promising technique for detecting circulating cancer biomarkers with greater sensitivity and reliability. Among the various circulating biomolecules, oncomiRs are emerging as prominent biomarkers for the early screening of breast cancers (BCs). In this review, we provide a comprehensive understanding of different SERS-based biosensors and their application to identify BC-specific oncomiRs. We also discuss different SERS-based sensing strategies, nano-analytical frameworks, and challenges to be addressed for effective clinical translation.

A photonic dual nano-hybrid assay for detection of cell-free circulating mitochondrial DNA.

Circulating cell free mitochondrial DNA (ccf-mtDNA) has emerged as a potential marker for diagnosis and prognosis of different chronic and age associated non-communicable diseases. Therefore, owing to its biomarker potential, we herein assessed a novel nano-photonic dual hybrid assay system for rapid and specific detection of ccf-mtDNA. The assay comprised of two systems, i.e. a capture and screen facet containing aminopyrene tethered carbon quantum dots for effective screening of circulating cell free nucleic acids (ccf-NAs) and a quantum dot conjugated probe for precise detection of ccf-mtDNA in the screened ccf-NAs. Our observations suggested that the developed dual-assay system possesses high feasibility and selectivity in screening of ccf-NAs and estimation of ccfmtDNA in a given sample. It also offers high versatility of measurement in different analytical platforms, indicating the translational potential of the method for possible disease risk assessment in control and field settings.

Gold based nano-photonic approach for point-of-care detection of circulating long non-coding RNAs.

Development of a rapid, sensitive and easy to use point of care assay for detection of circulating long non-coding RNAs (lncRNAs) is of great importance. These biomolecules possess the ability to regulate vital cellular processes and act as biomarkers for various human non-communicable diseases. The present work aimed to develop a simplified and reliable cytometric fluorescence-based approach for precise recognition of circulating lncRNAs in a given sample using biotinylated uracil-modified oligonucleotide tethered AlexaFluor488-labeled streptavidin gold colloidal (BiO-StrAG) nano-conjugates. The fluorophores in close proximity to the gold nanoparticles result in quenching of fluorescence; however, specific recognition of target lncRNAs increases this distance which causes plasmonic enhancement of fluorescence. As per the flow cytometry and fluorometry investigations, the developed methodology provides a precise and sensitive approach for detection of the target lncRNAs (up to 5 nM in any given sample). With advantages of high selectivity and feasibility, our strategy offers great potential of being developed as a promising tool for interrogating aberrant regulation of lncRNAs functions, especially indicated in various diseased states.

Mitochondrial-induced Epigenetic Modifications: From Biology to Clinical Translation.

Mitochondria are maternally inherited semi-autonomous organelles that play a central role in redox balance, energy metabolism, control of integrated stress responses, and cellular homeostasis. The molecular communication between mitochondria and the nucleus is intricate and bidirectional in nature. Though mitochondrial genome encodes for several key proteins involved in oxidative phosphorylation, several regulatory factors encoded by nuclear DNA are prominent contributors to mitochondrial biogenesis and function. The loss of synergy between this reciprocal control of anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signaling, triggers epigenomic imbalance and affects mitochondrial function and global gene expressions. Recent expansions of our knowledge on mitochondrial epigenomics have offered novel perspectives for the study of several non-communicable diseases including cancer. As mitochondria are considered beacons for pharmacological interventions, new frontiers in targeted delivery approaches could provide opportunities for effective disease management and cure through reversible epigenetic reprogramming. This review focuses on recent progress in the area of mitochondrial-nuclear cross-talk and epigenetic regulation of mitochondrial DNA methylation, mitochondrial micro RNAs, and post-translational modification of mitochondrial nucleoid-associated proteins that hold major opportunities for targeted drug delivery and clinical translation.

Immuno-cytometric detection of circulating cell free methylated DNA, post-translationally modified histones and micro RNAs using semi-conducting nanocrystals.

The diagnostic potential of cell free epigenomic signatures is largely driven by the fact that manifold quantities of methylated DNA, post-translationally modified histones and micro RNAs are released into systemic circulation in various non-communicable diseases. However, the time-consuming and specificity-related complications of conventional analytical procedures necessitate the development of a method which is rapid, selective and sensitive in nature. The present work illustrates a novel; prompt; "mix and measure" cytometric-based nano-biosensing system that offers direct quantification of cell-free circulating (ccf) epigenomic signatures (methylated ccf-DNA, tri-methylated histone H3 at lysine {4, 9, 27 & 36} and argonaute 2 protein-bound ccf-micro RNAs) using triple nano-assemblies in a single tube format. Each assembly with unique structural and spectral properties comprised of n-type semiconducting nanocrystals conjugated to a specific monoclonal antibody. Our results suggested that the developed combinatorial approach may offer simultaneous detection of three distinct yet biologically interrelated signatures with high selectivity and sensitivity using flow cytometry and fluorometry in the enriched and test samples. The proposed novel nano-assembly based detection system has a considerable potential of emerging as a minimal invasive easy-to-use method that could possibly permit real-time, rapid and reproducible monitoring of epigenomic markers in clinical and field settings.

Clostridium perfringens phospholipase C impairs innate immune response by inducing integrated stress response and mitochondrial-induced epigenetic modifications.

Clostridium perfringens, a rod-shaped, gram-positive, anaerobic, spore-forming bacterium is one of the most widely occurring bacterial pathogens, associated with a spectrum of diseases in humans. A major virulence factor during its infection is the enzyme phospholipase C encoded by the plc gene, known as Clostridium perfringens phospholipase C (CpPLC). The present study was designed to understand the role of CpPLC in inducing survival mechanisms and mitochondrial-induced epigenetic changes in a human lymphocyte cell culture model. Following exposure to CpPLC, a significant generation of mitochondrial reactive oxygen species was observed, which coincided with the changes in the expression of vital components of MAP/ERK/RTK signaling cascade that regulates the downstream cellular functions. These disturbances further led to alterations in the mitochondrial genome and functioning. This was supported by the observed upregulation in the expression of mitochondrial fission genes Drp1, Fis1, and Mff, and mitochondrial fusion genes MFN1, MFN2, and OPA1 following CpPLC exposure. CpPLC exposed cells showed upregulation of OMA1, DELE1, and HRI genes involved in the integrated stress response (ISR), which suggests that it may induce the ISR that provides a pro-survival mechanism to the host cell. CpPLC also initiated immune patho-physiologic mechanisms including mitochondrial-induced epigenetic modifications through a mitochondrial-ROS driven signaling pathway. Interestingly, epigenetic machinery not only play a pivotal role in lymphocyte homeostasis by contributing to cell-fate decisions but thought to be one of the mechanisms by which intracellular pathogens survive within the host cells. Importantly, the impairment of mtDNA repair among the CpPLC exposed cells, induced alterations within mtDNA methylation, and led to the deregulation of MT-CO1, MT-ND6, MT-ATPase 6, and MT-ATPase8 gene expression profiles that are important for mitochondrial bioenergetics and subsequent metabolic pathways. This was further confirmed by the changes in the activity of mitochondrial electron chain complexes (complex I, II, III, IV and V). The altered mtDNA methylation profile was also found to be closely associated with the varied expression of mitomiRs and their targets. CpPLC exposed cells showed up-regulation of miR24 expression and down-regulation of miR34a, miR150, and miR155, while the increased expression of mitomiR target genes i.e. of K-Ras, MYC, EGFR, and NF-kß was also observed in these cells. Altogether, our findings provide novel insights into the derailment of redox signaling machinery in CpPLC treated lymphocytes and its role in the induction of survival mechanisms and mitochondrial-induced epigenetic modifications.

Mapping the Mitochondrial Regulation of Epigenetic Modifications in Association With Carcinogenic and Noncarcinogenic Polycyclic Aromatic Hydrocarbon Exposure.

Polycyclic aromatic hydrocarbons (PAHs) refer to a ubiquitous group of anthropogenic air pollutants that are generated through incomplete carbon combustion. Although the immunotoxic nature of PAHs has been previously reported, the underlying molecular mechanisms of this effect are not fully understood. In the present study, we investigated the mitochondrial-mediated epigenetic regulation of 2 PAHs, carcinogenic (benzo[a]pyrene; BaP) and noncarcinogenic (anthracene [ANT]), in peripheral lymphocytes. While ANT exposure triggered mitochondrial oxidative damage, no appreciable epigenetic modifications were observed. On the other hand, exposure to BaP perturbed the mitochondrial redox machinery and initiated cascade of epigenetic modifications. Cells exposed to BaP showed prominent changes in the expression of mitochondrial microRNAs (miR-24, miR-34a, miR-150, and miR-155) and their respective gene targets (NF-κß, MYC, and p53). The exposure of BaP also caused significant alterations in the expression of epigenetic modifiers (DNMT1, HDAC1, HDAC7, KDM3a, EZH2, and P300) and hypomethylation within nuclear and mitochondrial DNA. This further induced methylation of histone tails, which play a crucial role in the regulation of chromatin structure. Overall, our study provides novel mechanistic insights into the mitochondrial regulation of epigenetic modifications in association with PAH-induced immunotoxicity.

Impairment of Mitochondrial-Nuclear Cross Talk in Lymphocytes Exposed to Landfill Leachate.

Landfill leachate, a complex mixture of different solid waste compounds, is widely known to possess toxic properties. However, the fundamental molecular mechanisms engaged with landfill leachate exposure inducing cellular and sub-cellular ramifications are not well explicated. Therefore, we aim to examine the potential of leachate to impair mitochondrial machinery and its associated mechanisms in human peripheral blood lymphocytes. On assessment, the significant increase in the dichlorofluorescein (DCF) fluorescence, accumulation of 8-Oxo-2'-deoxyguanosine (8-oxo-dG), and levels of nuclear factor erythroid 2-related factor 2 (Nrf-2) strongly indicated the ability of the leachate to induce a pro-oxidant state inside the cell. The decrease in the mitochondrial membrane potential and alterations in the mitochondrial genome observed in leachate-exposed cells further suggested the disturbances in mitochondrial machinery. Moreover, these mitochondrial-associated redox imbalances were accompanied by the increased level of NF-κß, pro-inflammatory cytokines, and DNA damage. In addition, the higher DNA fragmentation, release of nucleosomes, levels of polyadenosine diphosphate ADP-ribose polymerase (PARP), and activity of caspase-3 suggested the involvement of mitochondrial mediated apoptosis in leachate exposed cells. These observations were accompanied by the low proliferative index of the exposed cells. Conclusively, our results clearly indicate the ability of landfill leachate to disturb mitochondrial redox homeostasis, which might be a probable source for the immunotoxic consequences leading to plausible patho-physiological conditions in humans susceptible to such environmental exposures.

Exposure to ultrafine particulate matter induces NF-κß mediated epigenetic modifications.

Exposure to ultrafine particulate matter (PM0.1) is positively associated with the etiology of different acute and chronic disorders; however, the in-depth biological imprints that link these submicron particles with the disturbances in the epigenomic machinery are not well defined. Earlier, we showed that exposure to these particles causes significant disturbances in the mitochondrial machinery and triggers PI-3-kinase mediated DNA damage responses. In the present study, we aimed to further understand the epigenomic insights of the ultrafine PM exposure. The higher levels of intracellular reactive oxygen species and depleted Nrf-2 in ultrafine PM exposed cells reconfirmed its potential to induce oxidative stress. Importantly, the observed increase in the levels of NF-κß and associated cytokines among exposed cells suggested the activation of NF-κß mediated inflammatory loop which potentially serves as a platform for initiating epigenetic insinuations. This fact was strongly supported by the altered miRNA expression profile of the ultrafine PM exposed cells. These NF-κß induced miRNA alterations were also found to be associated with other epigenetic targets as the exposed cells showed higher expression levels of DNA methyltransferases which positively corresponded with the global changes in DNA methylation levels. Upon further analysis, significant alterations in histone code were also reported in ultrafine PM exposed cells. Conclusively our results suggested that NF-κß acts as an inflammatory switch that possesses the potential to induce genome-wide epigenetic modification upon ultrafine PM exposure.