Sunil Kumar Singh

Amine-Modified Graphene: Thrombo-Protective Safer Alternative to Graphene Oxide for Biomedical Applications

Graphene and its derivatives have attracted significant research interest based on their application potential in different fields including biomedicine. However, recent reports from our laboratory and elsewhere have pointed to serious toxic effects of this nanomaterial on cells and organisms. Graphene oxide (GO) was found to be highly thrombogenic in mouse and evoked strong aggregatory response in human platelets. As platelets play a central role in hemostasis and thrombus formation, thrombotoxicity of GO potentially limits its biomedical applications. Surface chemistry of nanomaterials is a critical determinant of biocompatibility, and thus differentially functionalized nanomaterials exhibit varied cellular toxicity. Amine-modified carbon nanotubes have recently been shown to possess cytoprotective action, which was not exhibited by their relatively toxic carboxylated counterparts. We, therefore, evaluated the effect of amine modification of graphene on platelet reactivity. Remarkably, our results revealed for the first time that amine-modified graphene (G-NH(2)) had absolutely no stimulatory effect on human platelets nor did it induce pulmonary thromboembolism in mice following intravenous administration. Further, it did not evoke lysis of erythrocytes, another major cellular component in blood. These findings contrasted strikingly the observations with GO and reduced GO (RGO). We conclude that G-NH(2) is not endowed with thrombotoxic property unlike other commonly investigated graphene derivatives and is thus potentially safe for in vivo biomedical applications.

Characterization of Antiplatelet Properties of Silver Nanoparticles

Thrombotic disorders have emerged as serious threat to society. As anticoagulant and thrombolytic therapies are usually associated with serious bleeding complications, the focus has now shifted to regulating and maintaining platelets in an inactive state. In the present study we show that nanosilver has an innate antiplatelet property and effectively prevents integrin-mediated platelet responses, both in vivo and in vitro, in a concentration-dependent manner. Ultrastructural studies show that nanosilver accumulates within platelet granules and reduces interplatelet proximity. Our findings further suggest that these nanoparticles do not confer any lytic effect on platelets and thus hold potential to be promoted as antiplatelet/antithrombotic agents after careful evaluation of toxic effects.

The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation

Arabidopsis IRX10 and IRX10-LIKE (IRX10-L) proteins are closely related members of the GT47 glycosyltransferase family. Single gene knock-outs of IRX10 or IRX10-L result in plants with either a weak or no mutant phenotype. However irx10 irx10-L double mutants are severely affected in their development, with a reduced rosette size and infrequent formation of a small infertile inflorescence. Plants homozygous for irx10 and heterozygous for irx10-L have an intermediate phenotype exhibiting a short inflorescence compared with the wild type, and an almost complete loss of fertility. Stem sections of the irx10 homozygous irx10-L heterozygous or irx10 irx10-L double mutants show decreased secondary cell-wall formation. NMR analysis shows that signals derived from the reducing end structure of glucuronoxylan were detected in the irx10 single mutant, and in the irx10 homozygous irx10-L heterozygous combination, but that the degree of polymerization of the xylan backbone was reduced compared with the wild type. Additionally, xylans from irx10 stem tissues have an almost complete loss of the GlcUA side chain, whereas the level of 4-O-Me-GlcUA was similar to that in wild type. Deletion of the predicted signal peptide from the N terminus of IRX10 or IRX10-L results in an inability to rescue the irx10 irx10-L double mutant phenotype. These findings demonstrate that IRX10 and IRX10-L perform a critical function in the synthesis of glucuronoxylan during secondary cell-wall formation, and that this activity is associated with the formation of the xylan backbone structure. This contrasts with the proposed function of the tobacco NpGUT1, which is closely related to the Arabidopsis IRX10 and IRX10-L proteins, in rhamnogalacturonan II biosynthesis.

Thrombus inducing property of atomically thin graphene oxide sheets

Graphene oxide (GO), the new two-dimensional carbon nanomaterial, is extensively investigated for potential biomedical applications. Thus, it is pertinent to critically evaluate its untoward effects on physiology of tissue systems including blood platelets, the cells responsible for maintenance of hemostasis and thrombus formation. Here we report for the first time that atomically thin GO sheets elicited strong aggregatory response in platelets through activation of Src kinases and release of calcium from intracellular stores. Compounding this, intravenous administration of GO was found to induce extensive pulmonary thromboembolism in mice. Prothrombotic character of GO was dependent on surface charge distribution as reduced GO (RGO) was significantly less effective in aggregating platelets. Our findings raise a concern on putative biomedical applications of GO in the form of diagnostic and therapeutic tools where its prothrombotic property should be carefully investigated.

AXR4 Is Required for Localization of the Auxin Influx Facilitator AUX1

The AUX1 and PIN auxin influx and efflux facilitators are key regulators of root growth and development. For root gravitropism to occur, AUX1 and PIN2 must transport auxin via the lateral root cap to elongating epidermal cells. Genetic studies suggest that AXR4 functions in the same pathway as AUX1. Here we show that AXR4 is a previously unidentified accessory protein of the endoplasmic reticulum (ER) that regulates localization of AUX1 but not of PIN proteins. Loss of AXR4 resulted in abnormal accumulation of AUX1 in the ER of epidermal cells, indicating that the axr4 agravitropic phenotype is caused by defective AUX1 trafficking in the root epidermis.

ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death.

Cell size and secondary cell wall patterning are crucial for the proper functioning of xylem vessel elements in the vascular tissues of plants. Through detailed anatomical characterization of Arabidopsis thaliana hypocotyls, we observed that mutations in the putative spermine biosynthetic gene ACL5 severely affected xylem specification: the xylem vessel elements of the acl5 mutant were small and mainly of the spiral type, and the normally predominant pitted vessels as well as the xylem fibers were completely missing. The cell-specific expression of ACL5 in the early developing vessel elements, as detected by in situ hybridization and reporter gene analyses, suggested that the observed xylem vessel defects were caused directly by the acl5 mutation. Exogenous spermine prolonged xylem element differentiation and stimulated cell expansion and cell wall elaboration in xylogenic cell cultures of Zinnia elegans, suggesting that ACL5 prevents premature death of the developing vessel elements to allow complete expansion and secondary cell wall patterning. This was further supported by our observations that the vessel elements of acl5 seemed to initiate the cell death program too early and that the xylem defects associated with acl5 could be largely phenocopied by induction of premature, diphtheria toxin-mediated cell death in the ACL5-expressing vessel elements. We therefore provide, for the first time, mechanistic evidence for the function of ACL5 in xylem specification through its action on the duration of xylem element differentiation.

Role of polyamines in plant vascular development.

Several pieces of evidence suggest a role for polyamines in the regulation of plant vascular development. For instance, polyamine oxidase gene expression has been shown to be associated with lignification, and downregulation of S-adenosylmethionine decarboxylase causes dwarfism and enlargement of the vasculature. Recent evidence from Arabidopsis thaliana also suggests that the active polyamine in the regulation of vascular development is the tetraamine thermospermine. Thermospermine biosynthesis is catalyzed by the aminopropyl transferase encoded by ACAULIS5, which is specifically expressed in xylem vessel elements. Both genetic and molecular evidence support a fundamental role for thermospermine in preventing premature maturation and death of the xylem vessel elements. This safeguard action of thermospermine has significant impact on xylem cell morphology, cell wall patterning and cell death as well as on plant growth in general. This manuscript reviews recent reports on polyamine function and places polyamines in the context of the known regulatory mechanisms that govern vascular development.

Insight into the early steps of root hair formation revealed by the procuste1 cellulose synthase mutant of Arabidopsis thaliana

BackgroundFormation of plant root hairs originating from epidermal cells involves selection of a polar initiation site and production of an initial hair bulge which requires local cell wall loosening. In Arabidopsis the polar initiation site is located towards the basal end of epidermal cells. However little is currently understood about the mechanism for the selection of the hair initiation site or the mechanism by which localised hair outgrowth is achieved. The Arabidopsis procuste1 (prc1-1) cellulose synthase mutant was studied in order to investigate the role of the cell wall loosening during the early stages of hair formation.ResultsThe prc1-1 mutant exhibits uncontrolled, preferential bulging of trichoblast cells coupled with mislocalised hair positioning. Combining the prc1-1 mutant with root hair defective6-1 (rhd6-1), which on its own is almost completely devoid of root hairs results in a significant restoration of root hair formation. The pEXPANSIN7::GFP (pEXP7::GFP) marker which is specifically expressed in trichoblast cell files of wild-type roots, is absent in the rhd6-1 mutant. However, pEXP7::GFP expression in the rhd6-1/prc1-1 double mutant is restored in a subset of epidermal cells which have either formed a root hair or exhibit a bulged phenotype consistent with a function for EXP7 during the early stages of hair formation.ConclusionThese results show that RHD6 acts upstream of the normal cell wall loosening event which involves EXP7 expression and that in the absence of a functional RHD6 the loosening and accompanying EXP7 expression is blocked. In the prc1-1 mutant background, the requirement for RHD6 during hair initiation is reduced which may result from a weaker cell wall structure mimicking the cell wall loosening events during hair formation.

Nanodiamonds activate blood platelets and induce thromboembolism

AIMnNanodiamonds (NDs) have been evaluated for a wide range of biomedical applications. Thus, thorough investigation of the biocompatibility of NDs has become a research priority. Platelets are highly sensitive and are one of the most abundant cell types found in blood. They have a central role in hemostasis and arterial thrombosis. In this study, we aim to investigate the direct and acute effects of carboxylated NDs on platelet function.nnnMETHODSnIn this study, pro-coagulant parameters such as platelet aggregability, intracellular Ca(2+) flux, mitochondrial transmembrane potential (ΔΨm), generation of reactive oxygen species, surface exposure of phosphatidylserine, electron microscopy, cell viability assay and in vivo thromboembolism were analyzed in great detail.nnnRESULTSnCarboxylated NDs evoked significant activation of human platelets. When administered intravenously in mice, NDs were found to induce widespread pulmonary thromboembolism, indicating the remarkable thrombogenic potential of this nanomaterial.nnnCONCLUSIONnOur findings raise concerns regarding the putative biomedical applications of NDs pertaining to diagnostics and therapeutics, and their toxicity and prothrombotic properties should be critically evaluated.

Novel microfilaricidal activity of nanosilver

Purpose The currently available drug repertoire against lymphatic filariasis, a major health hazard in the developing world, is inadequate and is fraught with serious limitations. Thus, the development of an effective antifilarial strategy has become a global research thrust mandated by the World Health Organization. Nanoparticles of silver endowed with antibacterial potency are known to induce apoptosis in eukaryotic cells. The present study was designed to investigate the possible microfilaricidal efficacy of silver nanoparticles and to establish the validity of apoptotic rationale in antifilarial drug designing. Methods This report analyzed the effect of nanoparticles of silver as well as gold (size range: 10–15 nm) on the microfilariae of Brugia malayi obtained from the lavage of peritoneal cavities of infected jirds (Meriones unguiculatus). The study included a microfilarial motility assay, a trypan blue exclusion test, a poly(adenosine diphosphate-ribose) polymerase activity study, ethidium bromide/acridine orange differential staining, and transmission, as well as scanning electron microscopic evaluation of ultrastructural changes in microfilariae. Results The study demonstrates that nanoparticles of silver, but not of gold, elicited significant loss in microfilarial motility. Differential staining of parasites with ethidium bromide and acridine orange, poly(adenosine diphosphate-ribose) polymerase activity in microfilarial lysate, and electron microscopic findings underscored apoptotic death of parasites attributable to nanosilver. In a trypan blue exclusion test, the 50% lethal dose of nanosilver was measured to be 101.2 μM, which was higher than the recorded complete inhibitory concentration value (50.6 μM), thus supporting nanosilver as a potential drug candidate against lymphatic filariasis. Conclusion The present report provides the first ever conclusive proof in support of apoptosis as a novel stratagem in antifilarial drug designing and nanoscale silver as a valid lead in research on antifilarial therapeutics. The main embargo about the current drug diethylcarbamazine citrate is its empirical use without rationale. Effective microfilaricidal activity of nanosilver at relatively low concentrations as reported in this study, with evidence of the induction of apoptosis in microfilariae, projects nanosilver as a potential drug adjuvant against lymphatic filariasis. The much higher 50% lethal dose value of nanosilver compared to the complete inhibitory concentration value reported in this study argues in favor of a safe therapeutic window of this agent in its antifilarial efficacy.