Ultrasensitive detection of 5-hydroxymethylcytosine in genomic DNA using a graphene-based sensor modified with biotin and gold nanoparticles.

Ten-eleven translocation (TET) proteins orchestrate deoxyribonucleic acid (DNA) methylation-demethylation dynamics by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and are frequently inactivated in various cancers. Due to the significance of 5hmC as an epigenetic biomarker for cancer diagnosis, pathogenesis, and treatment, its rapid and precise quantification is essential. Here, we report a highly sensitive electrochemical method for quantifying genomic 5hmC using graphene sheets that were electrochemically exfoliated and functionalized with biotin and gold nanoparticles (Bt-AuNPs) through a single-step electrical method. The attachment of Bt-AuNPs to graphene enhances the specificity of 5hmC-containing DNA and augments the oxidation of 5hmC to 5-formylcytosine in DNA. When coupled to a gold electrode, the Bt-AuNP-graphene-based sensor exhibits exceptional sensitivity and specificity for detecting 5hmC, with a detection limit of 63.2 fM. Furthermore, our sensor exhibits a remarkable capacity to measure 5hmC levels across a range of biological samples, including preclinical mouse tissues with varying 5hmC levels due to either TET gene disruption or oncogenic transformation, as well as human prostate cancer cell lines. Therefore, our sensing strategy has substantial potential for cancer diagnostics and prognosis.

A fast and label-free detection of hydroxymethylated DNA using a nozzle-jet printed AuNPs@Ti3C2 MXene-based electrochemical sensor.

5-hydroxymethylcytosine (5hmC) is a key epigenetic mark in the mammalian genome that has been proposed as a promising cancer biomarker with diagnostic and prognostic potentials. A new type of two-dimensional (2D) material called MXene includes transition metal carbides and nitrides and possesses unique physico-chemical properties suitable for diverse applications, including electrochemical sensors. Here, we report a new nozzle-jet printed electrochemical sensor using gold nanoparticles (AuNPs)@Ti3C2 MXene nanocomposite for the real-time and label-free detection of 5hmC in the genome. We utilized Ti3C2 MXene as a platform to immobilize AuNPs, which have been shown to exhibit different affinity interactions toward 5-methylcytosine (5 mC) and 5hmC, and thus produce distinct electrochemical responses. To fabricate the electrode, a highly conductive and adhesive silver ink was prepared to generate a silver line onto polyethylene terephthalate (PET) substrate using nozzle-jet printing, followed by deposition of AuNPs@Ti3C2 MXene ink at one end via dropcasting. Analyses of morphology and chemical composition showed that all steps of the sensor fabrication were successful. The fabricated sensor coupled with cyclic voltammetry showed excellent performance in distinguishing 5 mC- or 5hmC-enriched cellular genomic DNAs. As a proof-of-concept investigation, we confirmed that our sensor readily and consistently detected 5hmC diminution in multiple tumors, compared to the paired normal tissues. Thus, our simple and cost-effective sensing strategy using printable AuNPs@Ti3C2 MXene ink holds promise for a wide range of practical applications in epigenetic studies as well as clinical settings.

Rapid and Label-Free Detection of 5-Hydroxymethylcytosine in Genomic DNA Using an Au/ZnO Nanorods Hybrid Nanostructure-Based Electrochemical Sensor.

Ten-eleven-translocation (TET) proteins modify DNA methylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss of 5hmC, a widely accepted epigenetic hallmark of cancers, is proposed as a biomarker for early cancer diagnosis and prognosis. Thus, precise quantification of 5hmC holds great potential for diverse clinical applications. DNAs containing 5mC or 5hmC display different adsorption affinity toward the gold surface, thus producing different electrochemical responses. Here a novel, label-free electrochemical sensor based on gold nanoparticles (Au NPs)/zinc oxide nanorods (ZnO NRs) nanostructure for the facile and real-time detection of 5hmC-enriched DNAs is reported. The hybrid structure is fabricated by the vertical hydrothermal growth of ZnO NRs onto indium tin oxide glass substrate, followed by the decoration of ZnO NRs with Au NPs via sputtering. Successful fabrication is confirmed by analyzing the morphology and chemical composition of the sensor. By coupling the fabricated sensor with cyclic voltammetry, its functionality in distinguishing genomic DNAs containing different levels of 5hmC is validated. Notably, the sensor device successfully and consistently detects 5hmC loss in primary hepatocellular carcinoma, compared to the normal tissues. Thus, the novel sensing strategy to assess DNA hydroxymethylation will likely find broad applications in early cancer diagnosis and prognosis evaluation.

Molecular approaches to lung cancer prevention.

Lung cancer is generally diagnosed at advanced stages when surgical resection is not possible. Late diagnosis, along with development of chemoresistance, results in high mortality. Preventive approaches, including smoking cessation, chemoprevention and early detection are needed to improve survival. Smoking cessation combined with low-dose computed tomography screening has modestly improved survival. Chemoprevention has also shown some promise. Despite these successes, most lung cancer cases remain undetected until advanced stages. Additional early detection strategies may further improve survival and treatment outcome. Molecular alterations taking place during lung carcinogenesis have the potential to be used in early detection via noninvasive methods and may also serve as biomarkers for success of chemopreventive approaches. This review focuses on the utilization of molecular biomarkers to increase the efficacy of various preventive approaches.

Recent Nano-based Therapeutic Intervention of Bioactive Sesquiterpenes: Prospects in Cancer Therapeutics.

In the recent scenario, nanotechnology-based therapeutics intervention has gained tremendous impetus all across the globe. Nano-based pharmacological intervention of various bioactive compounds has been explored on an increasing scale. Sesquiterpenes are major constituents of essential oils (EOs) present in various plant species which possess intriguing therapeutic potentials. However, owing to their poor physicochemical properties; they have pharmacological limitations. Recent advances in nano-based therapeutic interventions offer various avenues to improve their therapeutic applicability. Reckoning with these, the present review collates various nano-based therapeutic intervention of sesquiterpenes with prospective potential against various debilitating diseases especially cancer. In our viewpoint, considering the burgeoning advancement in the field of nanomedicine; in the near future, the clinical applicability of these nano-formulated sesquiterpenes can be foreseen with great enthusiasm.

Adeno-Associated Virus as an Effective Malaria Booster Vaccine Following Adenovirus Priming.

An ideal malaria vaccine platform should potently induce protective immune responses and block parasite transmission from mosquito to human, and it should maintain these effects for an extended period. Here, we have focused on vaccine development based on adeno-associated virus serotype 1 (AAV1), a viral vector widely studied in the field of clinical gene therapy that is able to induce long-term transgene expression without causing toxicity in vivo. Our results show the potential utility of AAV1 vectors as an extremely potent booster vaccine to induce durable immunity when combined with an adenovirus-priming vaccine in a rodent malaria model. We generated a series of recombinant AAV1s and human adenovirus type 5 (AdHu5) expressing either Plasmodium falciparum circumsporozoite protein (PfCSP) or P25 (Pfs25) protein. Heterologous two-dose immunization with an AdHu5-prime and AAV1-boost (AdHu5-AAV1) elicited robust and durable PfCSP- or Pfs25-specific functional antibodies over 280 days. Regarding protective efficacy, AdHu5-AAV1 PfCSP achieved high sterile protection (up to 80% protection rate) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. When examining transmission-blocking (TB) efficacy, we found that immunization with AdHu5-AAV1 Pfs25 maintained TB activity in vivo against transgenic P. berghei expressing Pfs25 for 287 days (99% reduction in oocyst intensity, 85% reduction in oocyst prevalence). Our data indicate that AAV1-based malaria vaccines can confer potent and durable protection as well as TB efficacy when administered following an AdHu5 priming vaccine, supporting the further evaluation of this regimen in clinical trials as a next-generation malaria vaccine platform.

Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium.

Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-α and IFN-γ were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-α neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.

Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin, a multifunctional protein.

Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.