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.

Fabrication of Enzyme-Free and Rapid Electrochemical Detection of Glucose Sensor Based on ZnO Rod and Ru Doped Carbon Nitride Modified Gold Transducer.

Over 3 in 4 adults with diabetes live in low- and middle-income counties and health expenditure also increased 316% over the last 15 years. In this regard, we fabricate low cost, reusable and rapid detection of diabetes sensor based on zinc oxide rod inserted ruthenium-doped carbon nitride (ZnO-g-Ru-C3N4) modified sensor device. Developed sensor device physically and electrochemically characterized using X-ray diffraction (XRD), fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), chronoamperometry (CA) and differential pulse voltammetry (DPV). Sensing device as an effective enzyme-free glucose detection with high sensitivity (346 µA/mM/cm2) over the applied lower potential of +0.26 V (vs. Ag/AgCl), fast response (3 s) and broad linear range of (2-28) mM, coupled with a lower limit of detection (3.5 nM). The biosensing device gives better anti-interference ability with justifiable reproducibility, reusability (single electrode re-use 26 times in physiological buffer and 3 times in serum) and stability. Moreover, the real-time applicability of the sensor device was evaluated in human blood, serum and urine samples.

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.

Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets.

Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets.

Bioinformatic Identification of Peptidomimetic-Based Inhibitors against Plasmodium falciparum Antigen AMA1.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a valuable vaccine candidate and exported on the merozoite surface at the time of erythrocyte invasion. PfAMA1 interacts with rhoptry neck protein PfRON2, a component of the rhoptry protein complex, which forms the tight junction at the time of invasion. Phage display studies have identified a 15-residue (F1) and a 20-residue (R1) peptide that bind to PfAMA1 and block the invasion of erythrocytes. Cocrystal structures of central region of PfAMA1 containing disulfide-linked clusters (domains I and II) with R1 peptide and a peptide derived from PfRON2 showed strong structural similarity in binding. The peptides bound to a hydrophobic groove surrounded by domain I and II loops. In this study, peptidomimetics based on the crucial PfAMA1-binding residues of PfRON2 peptide have been identified. Top 5 peptidomimetics when checked for their docking on the region of PfAMA1 encompassing the hydrophobic groove were found to dock on the groove. Drug-like molecules having structural similarity to the top 5 peptidomimetics were identified based on their binding ability to PfAMA1 hydrophobic groove in blind docking. These inhibitors provide potential lead compounds, which could be used in the development of antimalarials targeting PfAMA1.

Exploiting unique structural and functional properties of malarial glycolytic enzymes for antimalarial drug development.

Metabolic enzymes have been known to carry out a variety of functions besides their normal housekeeping roles known as "moonlighting functions." These functionalities arise from structural changes induced by posttranslational modifications and/or binding of interacting proteins. Glycolysis is the sole source of energy generation for malaria parasite Plasmodium falciparum, hence a potential pathway for therapeutic intervention. Crystal structures of several P. falciparum glycolytic enzymes have been solved, revealing that they exhibit unique structural differences from the respective host enzymes, which could be exploited for their selective targeting. In addition, these enzymes carry out many parasite-specific functions, which could be of potential interest to control parasite development and transmission. This review focuses on the moonlighting functions of P. falciparum glycolytic enzymes and unique structural differences and functional features of the parasite enzymes, which could be exploited for therapeutic and transmission blocking interventions against malaria.

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.

Inhibitory potential of prodomain of Plasmodium falciparum protease serine repeat antigen 5 for asexual blood stages of parasite.

Plasmodium falciparum serine repeat antigen 5 (SERA5) is a target for both drug and vaccine intervention against malaria. SERA5 is secreted in the parasitophorous vacuole where it is proteolytically processed before schizont rupture. Among the processed products is a 50.8-kDa central domain of the protease, which possesses chymotrypsin-like activity and consists of a 28.9-kDa catalytic domain with a 21.9-kDa N-terminal prodomain, which remain attached together. Because SERA5 has been implicated in merozoite egress from host erythrocytes, the effect of the prodomain and a heptapeptide derived from its C-terminus spanning from D(560) to F(566) (DNSDNMF) on parasite growth was studied. When E. coli-expressed prodomain was incubated with parasite culture, a significant delay in transition from schizont to ring stages was observed up to nanomolar concentrations. The peptide, DNSDNMF also showed similar effects but at nearly 1000-fold higher concentrations. The peptide was also found to interact with the catalytic domain. These data demonstrate the crucial role of SERA5 prodomain for the egress process. Given the inhibitory potential of the prodomain for the parasite, we suggest that peptidomimetic inhibitors based on SERA5 prodomain sequences can be developed as future therapeutics against malaria.

Expression and characterization of catalytic domain of Plasmodium falciparum subtilisin-like protease 3.

PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database "PlasmoDB". The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn(334) to Glu(769) (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr(516) to Glu(769) (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes.