Detection of methylation on dsDNA using nanopores in a MoS2 membrane.

Methylation at the 5-carbon position of the cytosine nucleotide base in DNA has been shown to be a reliable diagnostic biomarker for carcinogenesis. Early detection of methylation and intervention could drastically increase the effectiveness of therapy and reduce the cancer mortality rate. Current methods for detecting methylation involve bisulfite genomic sequencing, which are cumbersome and demand a large sample size of bodily fluids to yield accurate results. Hence, more efficient and cost effective methods are desired. Based on our previous work, we present a novel nanopore-based assay using a nanopore in a MoS2 membrane, and the methyl-binding protein (MBP), MBD1x, to detect methylation on dsDNA. We show that the dsDNA translocation was effectively slowed down using an asymmetric concentration of buffer and explore the possibility of profiling the position of methylcytosines on the DNA strands as they translocate through the 2D membrane. Our findings advance us one step closer towards the possible use of nanopore sensing technology in medical applications such as cancer detection.

Tip-Based Nanofabrication of Arbitrary Shapes of Graphene Nanoribbons for Device Applications.

Graphene nanoribbons (GNRs) have promising applications in future nanoelectronics, chemical sensing and electrical interconnects. Although there are quite a few GNR nanofabrication methods reported, a rapid and low-cost fabrication method that is capable of fabricating arbitrary shapes of GNRs with good-quality is still in demand for using GNRs for device applications. In this paper, we present a tip-based nanofabrication method capable of fabricating arbitrary shapes of GNRs. A heated atomic force microscope (AFM) tip deposits polymer nanowires atop a CVD-grown graphene surface. The polymer nanowires serve as an etch mask to define GNRs through one step of oxygen plasma etching similar to a photoresist in conventional photolithography. Various shapes of GNRs with either linear or curvilinear features are demonstrated. The width of the GNR is around 270 nm and is determined by the width of the depositing polymer nanowire, which we estimate can be scaled down 15 nms. We characterize our TBN-fabricated GNRs using Raman spectroscopy and I-V measurements. The measured sheet resistances of our GNRs fall within the range of 1.65 kΩ/□-1 - 2.64 kΩ/□-1, in agreement with previously reported values. Furthermore, we determined the high-field breakdown current density of GNRs to be approximately 2.94×108 A/cm2. This TBN process is seamlessly compatible with existing nanofabrication processes, and is particularly suitable for fabricating GNR based electronic devices including next generation DNA sequencing technologies and beyond silicon field effect transistors.

Slowing DNA Transport Using Graphene-DNA Interactions.

Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases.

Nuclear anomalies, chromosomal aberrations and proliferation rates in cultured lymphocytes of head and neck cancer patients.

Head and neck cancers (HNC) are extremely complex disease types and it is likely that chromosomal instability is involved in the genetic mechanisms of its genesis. However, there is little information regarding the background levels of chromosome instability in these patients. In this pilot study, we examined spontaneous chromosome instability in short-term lymphocyte cultures (72 hours) from 72 study subjects - 36 newly diagnosed HNC squamous cell carcinoma patients and 36 healthy ethnic controls. We estimated chromosome instability (CIN) using chromosomal aberration (CA) analysis and nuclear level anomalies using the Cytokinesis Block Micronucleus Cytome Assay (CBMN Cyt Assay). The proliferation rates in cultures of peripheral blood lymphocytes (PBL) were assessed by calculating the Cytokinesis Block Proliferation Index (CBPI). Our results showed a significantly higher mean level of spontaneous chromosome type aberrations (CSAs), chromatid type aberration (CTAs) dicentric chromosomes (DIC) and chromosome aneuploidy (CANEUP) in patients (CSAs, 0.0294±0.0038; CTAs, 0.0925±0.0060; DICs, 0.0213±0.0028; and CANEUPs, 0.0308±0.0035) compared to controls (CSAs, 0.0005±0.0003; CTAs, 0.0058±0.0015; DICs, 0.0005±0.0003; and CANEUPs, 0.0052±0.0013) where p<0.001. Similarly, spontaneous nuclear anomalies showed significantly higher mean level of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) among cases (MNi, 0.01867±0.00108; NPBs, 0.01561±0.00234; NBUDs, 0.00658±0.00068) compared with controls (MNi, 0.00027±0.00009; NPBs, 0.00002±0.00002; NBUDs, 0.00011±0.00007).The evaluation of CBPI supported genomic instability in the peripheral blood lymphocytes showing a significantly lower proliferation rate in HNC patients (1.525±0.005552) compared to healthy subjects (1.686±0.009520 ) (p<0.0001). In conclusion, our preliminary results showed that visible spontaneous genomic instability and low rate proliferation in the cultured peripheral lymphocytes of solid tumors could be biomarkers to predict malignancy in early stages.

Electrochemistry at the edge of a single graphene layer in a nanopore.

We study the electrochemistry of single layer graphene edges using a nanopore-based structure consisting of stacked graphene and Al(2)O(3) dielectric layers. Nanopores, with diameters ranging from 5 to 20 nm, are formed by an electron beam sculpting process on the stacked layers. This leads to a unique edge structure which, along with the atomically thin nature of the embedded graphene electrode, demonstrates electrochemical current densities as high as 1.2 × 10(4) A/cm(2). The graphene edge embedded structure offers a unique capability to study the electrochemical exchange at an individual graphene edge, isolated from the basal plane electrochemical activity. We also report ionic current modulation in the nanopore by biasing the embedded graphene terminal with respect to the electrodes in the fluid. The high electrochemical specific current density for a graphene nanopore-based device can have many applications in sensitive chemical and biological sensing, and energy storage devices.

Stacked graphene-Al2O3 nanopore sensors for sensitive detection of DNA and DNA-protein complexes.

We report the development of a multilayered graphene-Al(2)O(3) nanopore platform for the sensitive detection of DNA and DNA-protein complexes. Graphene-Al(2)O(3) nanolaminate membranes are formed by sequentially depositing layers of graphene and Al(2)O(3), with nanopores being formed in these membranes using an electron-beam sculpting process. The resulting nanopores are highly robust, exhibit low electrical noise (significantly lower than nanopores in pure graphene), are highly sensitive to electrolyte pH at low KCl concentrations (attributed to the high buffer capacity of Al(2)O(3)), and permit the electrical biasing of the embedded graphene electrode, thereby allowing for three terminal nanopore measurements. In proof-of-principle biomolecule sensing experiments, the folded and unfolded transport of single DNA molecules and RecA-coated DNA complexes could be discerned with high temporal resolution. The process described here also enables nanopore integration with new graphene-based structures, including nanoribbons and nanogaps, for single-molecule DNA sequencing and medical diagnostic applications.

Momordicatin purified from fruits of Momordica charantia is effective to act as a potent antileishmania agent.

Aqueous extract of the green fruits of the Indian plant Momordica charantia and purified Momordicatin structurally established as 4-(o-carboethoxyphenyl) butanol were evaluated in vitro and in vivo against kala-azar caused by Leishmania donovani. 50% inhibitory concentration (IC(50)) against Leishmania promastigotes in vitro for the crude extract and momordicatin were 0.6mg/L and 0.02mg/L, respectively. When administered in the hamster model of visceral leishmaniasis, 100% parasite clearance was achieved at a dose of 300mg/kg body weight of crude extract and 10mg/kg body weight of Momordicatin. Fe containing parasite superoxide dismutase (SOD) was totally inhibited when treated with 0.72mg/L crude extract and 0.20mg/L Momordicatin, respectively, whereas Cu-Zn containing SOD present in host remained unaffected. Results reveal that the mode of action of these newly found antileishmanial agents is mediated through inhibiting parasite SOD which is one of the key enzymes of the oxidative burst. It may be proposed from the present study that both crude extract of Momordica charantia and Momordicatin obtained from the fruits of the said plant may be considered as potential candidates towards developing new chemotherapeutics against leishmaniasis.

Peroxisome is a reservoir of intracellular calcium.

We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca(2+)-ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium.

An intracellular calcium store is present in Leishmania donovani glycosomes.

A fourth intracellular Ca2+ pool in Leishmania donovani was identified by permeabilizing plasma membrane with digitonin. In Fura 2 loaded cells Ca2+ was released synergistically when mitochondrial function was blocked by antimycin and oligomycin. Vanadate did not have any effect if applied before incorporation of these mitochondrial poisons. However, the same inhibitor which inhibits Ca2+-ATPase activity of endoplasmic reticulum was able to release Ca2+ at a slow rate when added after antimycin and oligomycin. Alkalization of cytoplasmic pH allowed further release of Ca2+ essentially from the acidocalcisome. Purified glycosomes could mediate Ca2+ uptake mechanism in presence of vanadate whereas bafilomycin, a specific and potent inhibitor of vacuolar proton pump did not have any effect. Glycosomal Ca2+-ATPase activity was optimum at pH 7.5. The apparent Km for calciumin presence of vanadate was 12 nM. Taken together, it may be suggested that a vanadate-insensitive Ca2+-ATPase is present in the membrane of this microbody. Presence of glycosomal Ca2+ was further confirmed by imaging of Ca2+ activity in the Fura 2 loaded purified organelle using confocal laser. Results reveal that newly localized glycosomal calcium may essentially be an effective candidate to play a significant role in cellular function.

Antiparasitic activity of a triphenyl tin complex against Leishmania donovani.

Visceral leishmaniasis is a life-threatening human disease commonly known as kala-azar. Leishmania donovani is the causative agent of this parasitic disease transmitted by the sand fly vector to infect hosts. Triphenyl tin salicylanilide thiosemicarbazone [Ph(3)Sn(OSal.TSCZH)] (TTST) which is an organometallic complex of triphenyl tin has been evaluated to explore possibility to develop a potent chemotherapeutic agent against visceral leishmaniasis. Effect of triphenyl tin complex on growth inhibition and host--parasite interaction were examined both in vitro and in vivo. Release of toxic superoxide radical was measured spectrophotometrically through the formation of blue formazan derived from reduced nitrobluetetrazolium. To understand mode of action of Ph(3)Sn(OSal.TSCZH), superoxide dismutase activity was determined spectrophotometrically by measuring ability of this enzyme to inhibit pyrogallol autoxidation and also by activity staining of the non-denaturing polyacrylamide gels after separating superoxide dismutase. Antileishmanial activity of triphenyl tin complex were found to be effective both in vitro and in vivo at lower concentrations compared to the existing toxic drugs available. IC(50) of Ph(3)Sn(OSal.TSCZH) was calculated as 0.05+/-0.01mg/L. Intracellular survival of the parasite in host macrophages was inhibited by TTST in a dose dependent manner. Parasite burden in spleen was reduced to 87% under TTST treatment (10mg/kg body weight) and under sodium antimony gluconate (20mg/kg body weight) reduced nearly to 65%. Its action as a chemotherapeutic agent is found to be mediated through inhibition of superoxide dismutase and simultaneous release of toxic superoxide radical. We propose that Ph(3) Sn(OSal.TSCZH) may be considered as a prospective candidate to establish a better line of therapeutic process against experimental visceral leishmaniasis.

Peroxisomal function is altered during leishmania infection.

BACKGROUND: The aim of this study was to investigate host peroxisomal properties after leishmania infection and provide clues to protect peroxisomes during the search for new chemotherapeutic strategies against leishmaniasis. MATERIAL/METHODS: To assess host peroxisomal properties after leishmania infection, we determined catalase, urate oxidase, superoxide dismutase (SOD), dihydroxyacetone phosphate acyl transferase (DHAPAT) and proteinase activities in peroxisomes purified by the nycodenz gradient technique. Spectrophotometric analysis of superoxide radical release and assays of palmitoyl Co-A oxidation were also conducted. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy studies were carried out to monitor the status of this microbody after parasite RESULTS: Liver peroxisomes were found to be functionally defective when purified after infection. The activities of catalase, urate oxidase, DHAPAT and SOD were either deficient or could not be detected after parasite infection. H2O2 producing peroxisomal b-oxidation was significantly elevated after 90 days of infection, with concomitant induction of superoxide radical production. Proteolytic activity in infected liver peroxisome was found to be inhibited, pointing to possible uneven processing of peroxisomal proteins. Electron microscopic studies revealed that the morphology of peroxisomes after leishmania infection was impaired. CONCLUSIONS: The evidence obtained for leishmania-induced peroxisomal dysfunction may provide clues to develop new drugs against this parasite, capable of protecting normal function of this ubiquitous host organelle for successful treatment.