[Show abstract][Hide abstract] ABSTRACT: Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called “tip cell,” followed by vessel elongation by “stalk cells.” Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40 % of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35 % more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.
[Show abstract][Hide abstract] ABSTRACT: We are describing immunochromatographic test strips with smart phone-based fluorescence readout. They are intended for use in the detection of the foodborne bacterial pathogens Salmonella spp. and Escherichia coli O157. Silica nanoparticles (SiNPs) were doped with FITC and Ru(bpy), conjugated to the respective antibodies, and then used in a conventional lateral flow immunoassay (LFIA). Fluorescence was recorded by inserting the nitrocellulose strip into a smart phone-based fluorimeter consisting of a light weight (40 g) optical module containing an LED light source, a fluorescence filter set and a lens attached to the integrated camera of the cell phone in order to acquire high-resolution fluorescence images. The images were analysed by exploiting the quick image processing application of the cell phone and enable the detection of pathogens within few minutes. This LFIA is capable of detecting pathogens in concentrations as low as 105 cfu mL−1 directly from test samples without pre-enrichment. The detection is one order of magnitude better compared to gold nanoparticle-based LFIAs under similar condition. The successful combination of fluorescent nanoparticle-based pathogen detection by LFIAs with a smart phone-based detection platform has resulted in a portable device with improved diagnosis features and having potential application in diagnostics and environmental monitoring.
The successful combination of fluorescent nanoparticle-based pathogen detection by lateral flow immunoassay with a smart phone-based detection platform has resulted in a portable device that enables rapid and reliable bacterial detection holding large potential in diagnostics and environmental monitoring
No preview · Article · Nov 2014 · Microchimica Acta
[Show abstract][Hide abstract] ABSTRACT: Homology modeling of the reductase domain of endothelial nitric oxide synthase (eNOS), which regulates the catalytic activity of eNOS, and molecular dynamics studies focusing especially on the serine residues S615, S633, and S1177 were performed. MD analysis of this structure revealed that S633 is highly flexible and accessible to solvent molecules, while S1177 becomes highly flexible when S633 is phosphorylated. The presence of intramolecular interactions between S1177 among the major serine residues underscores its structural importance to the efficient synthesis of nitric oxide in endothelium. In order to evaluate the appropriateness of phosphomimetic (for phosphorylation) and phosphomutant (for dephosphorylation) eNOSs for use as experimental model systems, the structural dynamics and conformational changes in phosphomimetic (S615D, S633D, S1177D) and phosphomutant (S615A, S633A, S1177A) eNOSs were investigated. Phosphomimetic and phosphomutant eNOSs portrayed S633 as a modulator of S1177, whereas such correlations could not be observed in native and phosphorylated eNOSs. Computational analysis of the docked complex revealed that phosphorylated pS1177 and pS615 have high affinity for Akt (one of the key kinases in the eNOS activation pathway), with a significant number of hydrogen bonds and salt bridges observed between these residues and Akt . This work therefore provides evidence of the subtle structural changes that occur within the reductase domain which contribute to the stability-flexibility-activity relationship of eNOS. Such subtle changes are of great importance in the context of regulated nitric oxide release by different phosphorylated forms of eNOS and the need to account for the existence of subtle differences between real proteins and experimental model systems.
Full-text · Article · Oct 2014 · Journal of Molecular Modeling
[Show abstract][Hide abstract] ABSTRACT: We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 104 cfu.mL−1 and 103 cfu.mL−1, respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods.
The method uses silica coated magnetic nanoparticles immobilized with anti-Salmonella antibody for immunomagnetic separation of Salmonella from beverages followed by detection by multiplex PCR (mPCR) and real time PCR (qPCR). This methodology contributes to rapid screening and accurate detection of Salmonella contaminations in beverages.
No preview · Article · Oct 2013 · Microchimica Acta
[Show abstract][Hide abstract] ABSTRACT: The vascular endothelium, the intima of blood vessels, coordinately interacts with several biochemical factors expressing endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO), a potent endogenous vasodilator. The present study investigated the regulation of eNOS by multiple molecular signal transduction pathways, namely vascular endothelial growth factor (VEGF-A) and shear stress which are implicated in the process of angiogenesis and vascular remodelling respectively. In response to signal transduction upstream by VEGF-A and shear stress, different signalling pathways mediated by kinases and intracellular calcium potentiates eNOS activation leading to nitric oxide release. Our study revealed a distinct pattern of eNOS activation driven by VEGF-A and shear stress, maintaining the signalling specificity of the respective pathways. A transient response to eNOS activation was observed under VEGF-A and shear stress stimulus when mediated by calcium dependent cascades, whereas a sustained response was produced by calcium independent vascular signalling kinases. Furthermore, we found that the basal arterial shear stress enhanced eNOS activity when stimulated synergistically even at low VEGF-A levels which might be utilized to facilitate specific endothelial cell functions. Moreover, our study revealed that the presence of PI3K imparted transient behaviour to PLCγ1 supporting the hypothesis that regression and formation of tube structures are mediated by PLCγ1 and PI3K respectively in endothelial cells. This fact is corroborated by the absence of transient behaviour when PI3K is inhibited. We therefore obtained subtle insights into the control mechanism governing the role of specific signalling proteins which are obligate for the regulation of endothelial cell function and the consequent modulation of the nitric oxide release pattern.
No preview · Article · Aug 2013 · Molecular BioSystems
[Show abstract][Hide abstract] ABSTRACT: Cadmium targets the vascular endothelium causing endothelial dysfunction and leakiness of endothelial barrier. Nitric oxide plays a major role in mediating endothelial functions including angiogenesis, migration and permeability. The present study investigates the nitric oxide effects on cadmium induced endothelial leakiness. Results of ex vivo and in vitro permeability assays showed that even a sub-lethal dose of cadmium chloride (1 µM) was sufficient to induce leakiness of endothelial cells. Cadmium drastically altered the actin polymerisation pattern and membrane tension of these cells compared to controls. Addition of nitric oxide donor Spermine NONOate (SP) significantly blunted cadmium-mediated effects and recover endothelial cells integrity. Cadmium-induced cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability and high reactive oxygen species generation. In brief, we show the protective role of nitric oxide against cadmium-mediated endothelial leakiness.
Full-text · Article · May 2013 · Cell Biology International
[Show abstract][Hide abstract] ABSTRACT: Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70 % and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2) cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100 % of E. coli, P. aeruginosa, P. mirabilis and 95 % of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3 days for detection.
No preview · Article · Apr 2012 · Current Microbiology
[Show abstract][Hide abstract] ABSTRACT: In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules.
We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains.
This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.
Preview · Article · Feb 2012 · Journal of Nanobiotechnology
[Show abstract][Hide abstract] ABSTRACT: Diamond-like nanocomposite (DLN) thin films are deposited on glass (pyrex/silicon) substrate by plasma enhanced chemical vapor deposition (PECVD) technique using different combinations of hexamethyldisiloxane (HMDSO) and hexamethyldisilazane (HMDSN) gas precursors. The surface morphology
of the DLN films has been investigated by atomic force microscopy (AFM). Nanoparticles on DLN films were analyzed by high-resolution transmission electron microscopy (HRTEM). DLN films contain Si3N4, SiC and SiO
nanoparticles (sizes ∼ 6 –30
nm) within amorphous matrix which reduce the compressive stress of the films and increase the adhesiveness between the films and substrates. The biocompatibility of the films is verified using cell adhesion and morphology studies of the DLN films. We have also observed the fluorescence emission
from the nanoparticles of the films. The biocompatibility of DLN films is examined by the cell viability test. This article emphasizes on the possible biomedical applications of prepared films like biosensors for diagnostics and therapies, surgical instruments, prosthetic replacements etc.
Chemically modified DLN surface can be act as a sensing trace of gases to detect biomolecules in biological research.
No preview · Article · Jan 2012 · Science of Advanced Materials
[Show abstract][Hide abstract] ABSTRACT: Abstract
A systems biology framework has been put forth to simulate the endothelial nitric oxide synthase (eNOS)
signaling in arterial endothelium. In this context we investigated one of the well known mechanical stimuli,
namely shear stress, that modulates vascular function in physiological and pathological conditions by triggering
complex network of intracellular signaling pathways. Since the kinetics of cellular response to shear stress
depends on various physiological factors regulating eNOS activity, a comprehensive simulation platform has
been developed using chemical kinetics approach underlying the role of two protein kinases, namely,
serine/threonine protein kinase (Akt) and protein kinase A (PKA). The in silico model developed validated the
activity of eNOS in cells that is regulated through a coordinated interaction of two protein kinase pathways. In
addition, we have deduced number of kinetic parameters that confer well with the experimental observations.
Further, we could infer the role of vascular signaling kinases that confer subtle variability in activation pattern of
eNOS. In the process, we have developed a systems simulation of eNOS signaling pathways that has implication
in shear stress mediated arterial contraction and relaxation processes.
Keywords: Nitric oxide synthase; shear stress; protein kinases; systems modeling; kinetic parameters;
No preview · Article · Dec 2011 · International Journal of Integrative Biology
[Show abstract][Hide abstract] ABSTRACT: We report a method to optically trap and micromanipulate metallic particles using IR laser. The experiment demonstrates the trapping of metallic particle using low NA objective lens (0.6 N.A). Unlike single beam gradient trapping of dielectric objects, the optical trapping of metallic particles occurs due to diffraction effect. We thus provide evidence for non-gradient forces playing a dominant role in the trapping of metallic particles, in here for the case of 3mum Fe particles, efficient trapping occurs at off-axis position (in the side lobes) of a focused laser beam. The optical trap is characterized by measuring the external magnetic field required to dislodge the Fe particle, and was found to be 0.03T to 0.11T for laser power 5 to 55mW at the sample.
Full-text · Article · Feb 2011 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: The composition of the cell membrane and the surrounding physiological factors determine the nature and dynamics of membrane-cytoskeleton coupling. Mechanical strength of a cell is mainly derived from such coupling. In this article, we investigate the effect of extra cellular cholesterol on the membrane-cytoskelaton connectivity of single cell endothelium and consequent remodeling of its mechanical properties. Using optical tweezers as a force probe, we have measured membrane stiffness (km), membrane microviscosity (εeff ) and the two-dimensional shear modulus (G'(f)) as a function of extracellular cholesterol in the range of 0.1mM to 6mM. We find that membrane stiffness and shear modulus are dependent on cholesterol-induced membrane-cytoskeletal organization. Further, by disrupting the membranecytoskeletal connectivity with Cytochalasin D, an actin delpolymerizing molecule, we recover pure membrane behaviour devoid of any cytoskeleton attachment. However, behaviour of εeff was found to be unaffected by disruption of membrane-cytoskeleton organization. We infer that cholesterol is playing a distinct role in modulating membrane organization and membrane-cytoskeleton connectivity independently. We further discuss implications of our approach in characterizing cellular mechanics.
No preview · Article · Aug 2010 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood flow. Endothelial cells (ECs) lining these blood vessels act as a sensing interface to transduce these hemodynamic forces or mechanical stimuli to intracellular signals. EC signaling leads to the production of nitric oxide (NO) that regulates the flow pressure through relaxation of smooth muscle. A theoretical frame work that captures the mechanics of vascular remodeling process along with intracellular signaling cascade can shed light in the process of adaptation and regulation of blood flow in these arteries. Towards this end, we have developed a detail mass action model of intracellular signaling, including shear induced eNOS activation, an enzyme that produces NO for signaling, leading to the regulation of NO production. We simulate shear-induced activation of eNOS in detail. We show that upon shear stimulus, concentration of GTP.G¿ increases instantly to 1.5 fold leading to increase in active eNOS to two fold. We further show that our approach is modular in that various pathways constituting the model of eNOS activation and smooth muscle relaxation can be included one-by-one.
[Show abstract][Hide abstract] ABSTRACT: Semiflexible biopolymers play a vital role in shaping cellular structure and rigidity. In this work, we report the determination
of microrheological properties of concentrated, double-stranded calf thymus DNA (CT-DNA) solutions using passive, laser-scattering
based particle-tracking methodology. From power spectral analysis, we obtain dynamic shear moduli of the polymer solutions
stretching over three decades of frequency (100–103 Hz) and over concentration ranges spanning from very dilute to concentrated regime. We also study the effects of altered
ionic strength and denaturation on the shear modulus. Our results indicate that (CT-DNA) exhibits predominantly elastic behaviour
in the concentration range we probed. From the measurements of the plateau shear modulus, G
p, we conclude that DNA generally behaves like a semiflexible polymer in a good solvent even at low ionic strength. We have
thus demonstrated application of passive microrheological method using optical tweezers to DNA solutions. Further extensions
of the technique and its applications are discussed.
Preview · Article · Jun 2008 · Bulletin of Materials Science
[Show abstract][Hide abstract] ABSTRACT: Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.
Full-text · Article · Apr 2008 · European Journal of Cell Biology